Soluble St2 Induces Cardiac Fibroblast Activation and Collagen Synthesis via Neuropilin-1

Circulating levels of soluble interleukin 1 receptor-like 1 (sST2) are increased in heart failure and associated with poor outcome, likely because of the activation of inflammation and fibrosis. We investigated the pathogenic role of sST2 as an inductor of cardiac fibroblasts activation and collagen synthesis. The effects of sST2 on human cardiac fibroblasts was assessed using proteomics and immunodetection approaches to evidence the upregulation of neuropilin-1 (NRP-1), a regulator of the profibrotic transforming growth factor (TGF)-β1. In parallel, sST2 increased fibroblast activation, collagen and fibrosis mediators. Pharmacological inhibition of nuclear factor-kappa B (NF-κB) restored NRP-1 levels and blocked profibrotic effects induced by sST2. In NRP-1 knockdown cells, sST2 failed to induce fibroblast activation and collagen synthesis. Exogenous NRP-1 enhanced cardiac fibroblast activation and collagen synthesis via NF-κB. In a pressure overload rat model, sST2 was elevated in association with cardiac fibrosis and was positively correlated with NRP-1 expression. Our study shows that sST2 induces human cardiac fibroblasts activation, as well as the synthesis of collagen and profibrotic molecules. These effects are mediated by NRP-1. The blockade of NF-κB restored NRP-1 expression, improving the profibrotic status induced by sST2. These results show a new pathogenic role for sST2 and its mediator, NRP-1, as cardiac fibroblast activators contributing to cardiac fibrosis.


Introduction
The interleukin 1 receptor-like 1 (IL1RL1) gene belongs to the IL-1 receptor family and encodes the protein ST2. The alternative splicing of IL1RL1 results in multiple transcript variants, including the membrane-bound ST2L receptor for IL-33 and a soluble inhibitory decoy receptor, sST2. Briefly, the IL-33/ST2L system is cardioprotective in both physiological and pathological conditions, promoting cell survival and blocking profibrotic intracellular signaling [1,2]. An imbalance of sST2 levels is one key contributor to cardiac fibrosis, which, finally, may lead to the development of heart failure (HF). Indeed, sST2 is produced by both cardiac fibroblasts and cardiomyocytes in response to injury or stress [3]. In cardiovascular cells, data concerning sST2 effects are scarce. Most of the studies have shown sST2 only as a decoy receptor that prevents IL-33/ST2L signaling. Thus, sST2 blocks the antihypertrophic effects of IL-33 in angiotensin II or phenylephrine-treated cardiomyocytes [3]. In endothelial cells, sST2 abolishes the increase in adhesion molecules and proinflammatory cytokines induced by IL-33 [4]. Noteworthy, sST2 can signal independently of IL-33 sequestration. In-line with this, in vascular smooth muscle cells, recombinant sST2 increases collagen, fibronectin, transforming growth factor (TGF)-β and connective tissue growth factor (CTGF) [5]. Moreover, sST2 promotes mitochondrial fusion in human cardiac fibroblasts (HCF), increasing oxidative stress and the secretion of inflammatory markers via NF-κB [6]. In clinical populations, higher levels of circulating sST2 are associated with increased myocardial fibrosis, adverse cardiac remodeling and worse cardiovascular outcomes [7]. However, the specific role of sST2 as a direct inductor of cardiac fibroblast activation and the synthesis of profibrotic molecules, to our knowledge, has never been investigated.
Cardiac fibroblasts are the major cells responsible for tissue fibrosis. Fibroblasts can be activated by a wide range of stimuli, TGF-β being one of the most-studied [8]. The conversion of quiescent fibroblasts into activated myofibroblasts promotes enhanced extracellular matrix production, a key event in cardiac fibrosis [9]. Furthermore, activated fibroblasts promote cardiomyocyte hypertrophy and dysfunction via the release of profibrotic factors, such as TGF-β1, generating a vicious circle [10]. However, the cell biology and molecular mechanisms of fibroblast activation remain poorly studied.
The aim of this study was to provide a mechanistic assessment of sST2 on human cardiac fibroblast activation into profibrotic myofibroblasts, using a proteomic approach, and to unravel the resulting in vitro effects in a pressure overload rat model.

Animal Model
Adult male Wistar rats (weight = 250-300 g) were obtained from Harlan Ibérica (Barcelona, Spain). Rats were randomly distributed into two different groups: control rats (control; n = 10) and rats subjected to pressure overload (PO) by placing a 0.58-mm (internal diameter) tantalum clip banding the ascending and supravalvular aorta, as previously described (n = 7) [11][12][13][14]. The occlusion of the aorta continued for six weeks. Then, the animals were finally euthanized [11]. Body weight was measured once a week. Systolic blood pressure was basally estimated as well after 3 weeks and at the end of the study by using tail-cuff plethysmographin unrestrained animals. The Animal Care and Use Committee of Universidad Complutense de Madrid approved all experimental procedures, according to the guidelines for ethical care of experimental animals of the European Community (approval ID: CEA-UCM 77/2012).

Mass Spectrometry Based-Quantitative Proteomics
Untreated cardiac fibroblasts and sST2-stimulated cardiac fibroblasts were compared by a shotgun comparative proteomic analysis using iTRAQ (isobaric Tags for Relative and Absolute Quantitation). Global experiments were carried out with whole cell lysates from four biological replicates (n = 4) in each experimental condition. Peptide labeling, peptide fractionation and mass spectrometry analysis were performed as previously described [15,16]. After MS/MS analysis, protein identification and relative quantification were performed with the ProteinPilot™ software (version 4.5; AB Sciex Spain S.L., Madrid, Spain) using the Paragon™ algorithm as the search engine. Although relative quantification and statistical analysis were provided by the ProteinPilot software, an additional 1.3-fold change cutoff for all iTRAQ ratios (ratio <0.77 or >1.3) and a p-value lower than 0.05 were selected to classify proteins as up-or downregulated (at least in two of three biological replicates). Proteins with iTRAQ ratios below the low range (0.77) were considered to be underexpressed, whereas those above the high range (1.3) were considered to be overexpressed. Proteomics validation was assayed in the same samples used for proteomics, and 5 additional biological replicates were included (n = 5). In addition, we assessed the expression of soluble NRP-1 in conditioned media randomly selected from different biological replicates. Each biological replicate was assayed at least in triplicate. Using a higher number of samples (at least 2 more HCF batches) was justified to avoid endowing statistical error type I, as no additional methods were used to assess sNRP-1.

CRISPR/Cas9 Genome Editing Mediated Deletion of NRP-1
NRP-1 expression was knocked down by CRISPR/Cas9 (clustered regularly interspaced short palindrome repeats) guided genome editing. Cells were seeded into 6-well plates at 70% confluence and transfected with a pool of three plasmids, each encoding the Cas9 nuclease and a target-specific 20-nt guide RNA (gRNA) designed for maximum gene editing efficiency according to the manufacturer's instructions (Santa Cruz Biotechnology, Heidelberg, Germany). Scramble gRNA CRISPR/Cas9 plasmid was used as a control.
Once NRP-1 knockout was generated, cells were treated with sST2 for 24 h. Then, cell extracts and supernatants were collected to evaluate fibroblast-to-myofibroblast transformations and the expression of fibrotic markers.

Real-Time Reverse Transcription PCR
Total RNA was extracted with Trizol Reagent (Qiagen, Hilden, Germany) and was reversetranscribed into single-stranded cDNA using a random hexamers master mix from Bio-Rad. Quantitative PCR (qPCR) analysis was performed using SYBR green PCR technology (Bio-Rad, Hercules, CA, USA) (Supplemental Table S1). Relative quantification was achieved with MyiQ software. HPRT, GADPH and β-actin were used as housekeeping genes for normalization. The relative expression (DDCT) of each selected gene product was calculated using the efficiency corrected calculation model and are shown as fold changes of the mRNA expression. All qPCRs were performed at least in triplicate for each experimental condition.

Statistical Analyses
Data are presented as scatter dot plots where horizontal line denotes a median value, and vertical lines denote the interquartile range (IQR). Normality of distributions was verified by means of the Kolmogorov-Smirnov test. The unpaired Student's t-test or the Mann Whitney U test were used to assess statistical differences between two experimental conditions as appropriate. Data were analyzed using a one-way analysis of variance, followed by Tukey's post hoc test analysis to assess specific differences among groups or conditions using GraphPad Software Inc (GraphPad, La Jolla, CA, USA). Pearson's coefficients were calculated to determine correlations. The predetermined significance level was p < 0.05.

sST2 Upregulates NRP-1 in Adult Human Cardiac Fibroblasts
A proteome-wide analysis of total cell extracts using isobaric tags (iTRAQ) coupled to 2D nanoliquid chromatography tandem mass spectrometry was performed in HCF treated with sST2 as previously reported [6]. NRP-1 was identified as an upregulated protein upon 24 h of stimulation (Supplemental Table S2). Complementary analyses validated the increase of NRP-1 protein levels induced by sST2 (p = 0.0079) (Figure 2A). Moreover, the stimulation with sST2 enhanced (p = 0.0125) the secretion of sNRP-1 ( Figure 2B). See the original Western blot images in Supplemental Figure 3.

Exogenous NRP-1 Increased the Expression of the Activation and Fibrosis Markers in Adult HCFs
In order to understand the contribution of sST2-induced sNRP-1 to the activation of profibrotic myofibroblast phenotypes, adult HCFs were treated with a soluble human recombinant NRP-1. Exogenous sNRP-1 was supplemented at different concentrations (10 −8 M, 10 −9 M and 10 −10 M) during 24 h to evidence the lowest concentration inducing myofibroblast activation. Exogenous recombinant NRP-1 increased the expression of the myofibroblast activation markers α-SMA and vimentin in a dose-dependent manner, reaching the statistical significance only for 10 −8 M (p = 0.0011 and p = 0.0288, respectively), with no effect on fibronectin secretion ( Figure 4A,B). Thereby, the 10 −8 M NRP-1 dose was subsequently used to explore the downstream signaling elicited by sNRP-1 on myofibroblast differentiation. Moreover, the treatment with NRP-1 enhanced collagen type I secretion also in a dose-dependent manner (p = 0.0379 for 10 −8 M) ( Figure 4C). Regarding other profibrotic markers, Gal-3 expression was increased with a dose-dependent pattern (p = 0.0006 for 10 −8 M) by sNRP-1 treatment ( Figure 4D), whereas CTGF expression was not modified ( Figure 4E). NRP-1-treated cells secreted similar levels of MMP-1, MMP-2, TIMP-1 or TIMP-2 as control cells ( Figure 4F). See original Western blot images in Supplemental Figures 6 and 7.

Cardiac Expression of sST2 and NRP-1 is Induced in a Pressure Overload Model
The expression of sST2 and sNRP-1 has been measured in myocardial tissues from a PO animal model. Briefly, PO rats presented increased cardiac fibrosis characterized by enhanced levels of α-SMA (1.6-fold), fibronectin (2.0-fold), collagen type I (2.3-fold), TGF-β (1.9-fold) and CTGF (1.7-fold) protein expressions (for further information, please refer to citation [11]). Perivascular myocardial fibrosis was also enhanced in PO rats. Moreover, α-SMA, vimentin and fibronectin immunostainings were increased in PO rats, as compared to the controls (Supplemental Figure S12). Interestingly, rats subjected to PO exhibited a greater expression of total ST2 and NRP-1 ((p = 0.0027 and p = 0.0068, respectively) ( Figure 6A,B). A specific transcript analysis suggests that ST2 elevation results from sST2 over-expression ( Figure 6C), while ST2L levels were not significantly different ( Figure 6D). Interestingly, ST2 was positively correlated with the cardiac expression of NRP-1 (r = 0.633, p = 0.02, Figure 6E). It is worth to note that the IL-33 expression was unaffected by the experimental condition both at the transcript ( Figure 6D) and protein levels ( Figure 6F). Intracellular pathways activated by the IL-33/ST2L pathway showed similar MyD88 levels and lower TRAF-6 and AP-1 expressions in PO rats, as compared to the controls ( Figure 6G). See original Western blot images in Supplemental Figure 11. . Effects of PO on IL-33 protein (F) and MyD88, TRAF-6 and AP-1 protein expressions (G). Scatter dot blot represents the median and minimum to a maximum range of N = 5-10 animals per group. For Western blot experiments, protein densitometry was expressed in arbitrary units (AU) once normalized to stain-free for proteins. Relative mRNA expression was expressed in arbitrary units (AU) of fold change after normalization to HPRT, β-actin and GADPH housekeeping genes. Representative blots have been displayed when appropriate. *p < 0.05 vs. control group. ST2, interleukin 1 receptor-like 1; NRP-1, neuropilin-1; IL-33, interleukin 33; MyD88, Myeloid differentiation primary response 88; TRAF-6, tumor necrosis factor receptor associated factor 6 and AP-1, activator protein 1.

Discussion
The purpose of this study was to investigate the effect of sST2 on fibroblast activation, collagen secretion and profibrotic marker productions in HCFs. sST2 enhanced the profibrotic myofibroblast differentiation, as demonstrated by the enhanced expression of fibrotic mediators and collagen type I secretion. Using a proteomic approach, NRP-1 has been identified as an upregulated protein by sST2. Our study shows that sST2 profibrotic effects are partly mediated by NRP-1, which emerges as a new inductor of fibroblast activations and a profibrotic phenotype in HCFs. In addition, we identified NF-κB as a central signaling pathway in the profibrotic effects of sST2 via NRP-1, and that was further validated in vivo.
Most of the sST2 previously defined effects suggest its function as a decoy receptor, inhibiting IL-33 effects [3,17,18]. However, sST2 could exert other effects independently of the sequestration of IL-33 [19]. In-line with this evidence, we previously described for the first time the deleterious effects triggered by sST2 on HCFs, by increasing the production of reactive oxygen species and inflammatory molecules [6]. In the present study, we expand our previous findings to show that sST2 also increased the myofibroblast activation markers, as well as collagen and other profibrotic markers, such as Gal-3, CTGF or TGF-β. Fibrosis is a fundamental component of the adverse structural remodeling of the myocardium present in a failing heart [20]. In HF patients, the sST2 measurement provides a strong serologic overview of the cumulative myocardial fibrotic process [21]. Our data reinforce the role of sST2 as a biomarker and, also, emerges as a biotarget of the fibrotic process, describing the mechanisms by which elevated sST2 could contribute to myofibroblast activation and collagen accumulation.
NRP-1 is a 130-kDa transmembrane protein identified as a coreceptor for multiple growth factors, including TGF-β1, where it could enhance responses to both the active and latent forms of this cytokine [22]. It is of special interest that the alternative splicing of mRNA encoding NRP-1 gives rise to several sNRP-1 (soluble NRP-1) isoforms [23]. sST2 enhanced both the membrane-bound and sNRP-1 in HCFs. This result was reinforced in the myocardium of PO rats, where increased ST2 levels correlated positively with the expression of NRP-1. Moreover, NRP-1 partly mediated the profibrotic sST2 effects in HCFs. Our data is in-line with other observations showing that NRP-1 could play a regulatory role in TGF-β1-induced fibrosis [24]. Interestingly, exogenous sNRP-1 induced myofibroblast activation, as well as collagen synthesis and the expression of profibrotic mediators. Although this is, to our knowledge, the first time that NRP-1 has been studied in HCFs, it has been previously suggested that NRP-1 promotes liver cirrhosis progression and its aggravation [25]. Moreover, the over-expression of NRP-1 promotes the endothelial-to-mesenchymal transition (EndMT) and associated fibrosis [26]. A positive correlation between NRP-1 levels, EndMT markers and profibrotic gene expressions has been reported in pancreatic ductal adenocarcinoma tissues [26]. In addition, the endothelial-specific loss of NRP-1 downregulates and inactivates TGF-β1 signaling, culminating in reduced CTGF and collagen type I [26]. Other results indicate that the NRP-1 antibody reduces fibrogenesis markers and inhibits fibrosis in hepatic stellate cells [22,25]. Thus, NRP-1 emerges as an attractive target in the context of fibrotic diseases.
NF-κB directly regulates the expression of fibrosis-related genes, including fibronectin and MMPs [27]. Moreover, NF-κB phosphorylation leads to the transformation of cardiac fibroblasts into myofibroblasts [28]. NF-κB phosphorylation also mediates the angiotensin II-stimulated profibrotic process [29] and regulates the levels of several profibrotic cytokines, leukocyte adhesion molecules and inflammatory molecules [30]. NF-κB has been previously described to contribute to the sST2 proinflammatory effects [6]. Our results, showing that NF-κB mediated the profibrotic effects induced by sST2, are not in agreement with a previous report showing that sST2 blocked IL-6 production by suppressing NF-κB activation in monocytic cells [31]. However, in the present study, both sST2 and NRP-1, a potential mediator of sST2 effects, did promote NF-κB phosphorylation in HCFs. Therefore, NF-κB seems to be a key regulator in controlling myofibroblast activation and collagen secretion elicited by both sST2 and NRP-1.

Conclusions
In summary, the present study shows that sST2 could affect myofibroblast activation, leading to an increase in collagen synthesis and profibrotic molecules in HCFs. Moreover, NRP-1, a molecule upregulated by sST2, emerges as a new, interesting target in cardiac fibrosis. Proinflammatory [6] and profibrotic effects triggered by sST2 via NF-KB certainly highlight the key role of the latter on cardiac fibrosis. New studies focusing on the interactions between sST2 and NRP-1 in experimental models are warranted.

Supplementary Materials:
The following are available online at www.mdpi.com/2073-4409/9/7/1667/s1: Figure  S1: original western blotting images for figure 1; Figure S2: original western blotting images for figure 1; Figure  S3: original western blotting images for figure 2; Figure S4: original western blotting images for figure 3; Figure  S5: original western blotting images for figure 3; Figure S6: original western blotting images for figure 4; Figure  S7: original western blotting images for figure 4; Figure S8: original western blotting images for figure 5; Figure  S9: original western blotting images for figure 5; Figure S10: original western blotting images for figure 5; Figure  S11: original western blotting images for figure 6; Figure S12: perivascular myocardial fibrosis and immunostainings in control and PO rats, Table S1: List of primers used for real-time PCR assays in the manuscript, Table S2: Proteome alterations induced by sST-2 in human cardiac fibroblasts.

Conflicts of Interest:
The authors state no conflicts of interest.