Role of sialidase Neu3 and ganglioside GM3 in cardiac fibroblasts activation

Cardiac ﬁ brosis is a key physiological response to cardiac tissue injury to protect the heart from wall rupture. However, its progression increases heart stiffness, eventually causing a decrease in heart contractility. Unfortunately, to date, no ef ﬁ cient anti ﬁ brotic therapies are available to the clinic. This is primarily due to the complexity of the process, which involves several cell types and signaling pathways. For instance, the transforming growth factor beta (TGF- β ) signaling pathway has been recognized to be vital for myo ﬁ broblasts activation and ﬁ brosis progression. In this context, complex sphingolipids, such as ganglioside GM3, have been shown to be directly involved in TGF- β receptor 1 (TGF-R1) activation. In this work, we report that an induced up-regulation of sialidase Neu3, a glycohydrolytic enzyme involved in ganglioside cell homeostasis, can signi ﬁ cantly reduce cardiac ﬁ brosis in primary cultures of human cardiac ﬁ broblasts by inhibiting the TGF- β signaling pathway, ultimately decreasing collagen I deposition. These results support the notion that modulating ganglioside GM3 cell content could represent a novel therapeutic approach for cardiac ﬁ brosis, warranting for further investigations.


Introduction
Fibrosis is a physiological process common to many organs, such as kidney [1], liver [2], lungs [3], and heart [4][5][6], which is characterized by the deposition of extracellular matrix (ECM) proteins in response to an injury [7]. Therefore, fibrosis is considered primarily as a reparative mechanism, since it promotes tissue healing. However, when not properly controlled, it could become pathologic, leading to parenchymal scarring, tissue remodeling and, eventually, to organ failure [8]. In this context, cardiac fibrosis has been defined as either reactive fibrosis or replacement fibrosis, depending on the stimuli [8]. Replacement fibrosis occurs after a massive loss of cardiomyocytes, as for example after myocardial infarction. Given the low regenerative capacity of the heart [9], the repair process aims to replace the dead cardiomyocytes with a fibrotic scar produced by activated fibroblasts. This response is fundamental, since it stabilizes ventricular walls, ultimately preventing their rupture [10]. However, its uncontrolled progression provokes chamber dilatation and hypertrophy, increases stiffness, and impairs electrical coupling, ultimately leading to heart failure [11]. Cardiac fibroblasts are the principal players of this mechanism and, upon appropriate stimuli, they can transdifferentiate into their active form, i.e. cardiac myofibroblasts [4]. Interestingly, cardiac fibroblasts are a peculiar cell type of embryonic epicardial and endothelial origins [12]. Their primary role is to furnish structural support for cardiomyocytes, regulating the homeostasis of the ECM [13]. Furthermore, they distribute mechanical forces and mediate electrical conduction [14]. After tissue injury, cardiomyocytes become apoptotic, endothelial cells modulate the inflammatory response, and proliferating immune cells infiltrate the damaged myocardium [15], causing an increase in inflammation and in profibrotic cytokines that activate cardiac fibroblasts. Differentiation towards myofibroblasts is characterized by the production of ECM proteins, such as collagen and fibronectin, and by the expression of stress fibers, composed mostly by α-smooth muscle actin (α-SMA), which is involved in the contractile activity [16]. The master regulator of fibrosis induction is TGF-β [17]: this cytokine binds to a heterodimeric membrane receptor composed of two subunits (TGF-β R1 and TGF-β R2), activating its intracellular canonical signaling cascade, including Smad family members [18]. The binding of TGF-β to its receptor induces the phosphorylation of the receptorregulated members of the Smad family (R-Smads), Smad2 and Smad3, which, in turn, interact with Smad4 and translocate as a complex to the nucleus, activating profibrotic gene expression [17]. TGF-β signaling is finely regulated, and the receptors represent the first step for its modulation [19]. Different factors could influence the activity of TGF-β receptors, such as proteins involved in ubiquitination [20], other receptors (i.e. endoglin [21] or ALK2/3/6 [22]), proteins involved in their trafficking towards the membranes (i.e. Rab GTPases [23], caveolin-1 [24]), or molecules that increase TGF-β response (i.e. ganglioside GM3 [25]). Among these factors, it has been demonstrated that ganglioside GM3 boosts the effects of TGF-β through the direct interaction with the TGF-β R1 in human lens epithelial cells, promoting the epithelial-to-mesenchymal transition [25]. Ganglioside GM3 is a member of the ganglioside family, which are glycosphingolipids containing sialic acid implicated in various biological processes, such as cell proliferation, cell interaction, differentiation, signal transduction, and stem cell markers [26][27][28]. GM3 could alter the activity of the membrane receptors of insulin, VEGF, EGF, or FGF; thus, it is implicated in different pathological processes, like obesity, insulin resistance, and tumor progression [29]. Its levels are tightly regulated by its synthesis by GM3 synthase [30], and by its degradation by Neu3 sialidase [31,32]. Sialidase Neu3 is a membrane glycosidase that removes sialic acid from GD1a and GM3 gangliosides [33] and is implicated in different cellular functions, including cell proliferation and differentiation [31,34]. We previously demonstrated that Neu3 is activated under hypoxic conditions both in vitro [35] and in vivo [36] and that its effects are mainly exerted by modulation of GM3 levels [31,33,35].
In this work, we assessed the effects of Neu3 overexpression on cardiac fibroblasts activation in a cellular model of cardiac fibrosis in order to identify new possible pharmacological targets for the development of new drugs for cardiac fibrosis modulation.

Ethical statement
All subjects gave their informed consent for inclusion before they participated in the study. The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by the Ethics Committee of the ASL MilanoDue (Protocol n. 2385).
Cardiac fibroblast isolation, culture, and stable overexpression of NEU3 Right atrial appendage biopsies were obtained from the point of atrial cannulation at the beginning of extracorporeal circulation. Tissue specimens weighed ∼100 mg and were collected in cold phosphate buffer solution (PBS) at pH 7.4, kept in ice, and processed within minutes after collection. Samples were washed with phosphate-buffered saline, cut into small pieces, and placed in the cell culture dish pre-coated with 1% porcine skin gelatin with 2 ml of growth medium composed of DMEM (Merck), with low glucose concentration (1 g/L), supplemented with 10% (v/v) fetal bovine serum (FBS) (Merck), 2 mM glutamine (Merck), and penicillin/ streptomycin 1X (Euroclone). Once the pieces attached to the plate, the growth medium was added up to 7 ml. The fibroblasts started to grow from the minced fragments in 2-3 days. When there were sufficient cells, they were detached enzymatically and plated in new dishes for proliferation. Cardiac fibroblasts stably overexpressing NEU3 sialidase were prepared with a lentiviral vector, according to our previously developed methods [37].

Fibroblasts activation
Cardiac fibroblasts were plated at 80-90% confluency and serum-starved for 48 h. Then, human recombinant TGF-β isoform 1 (Peprotech) was added to a final concentration of 10 ng/ml for 72 h.

Immunofluorescence
Cardiac fibroblasts were rinsed twice with PBS and then fixed using a solution of 4% paraformaldehyde in PBS for 15 min at room temperature. Fixed cells were washed with PBS three times for 5 min. Samples were then treated with blocking solution (PBS, 5% goat serum, Tween-20 0.1%) for 1 h at RT. Cells were then incubated with the primary antibody mouse monoclonal anti-α-Smooth muscle actin (α-SMA) (1 : 200, #A52228, Merck) for 2 h at RT. Cells were then washed with PBS, three times for 5 min, and incubated with secondary antibody (FITC goat anti-mouse, Jackson Laboratories; dilution 1 : 500 in blocking solution), 1 h at RT. Samples were finally washed with PBS (three times for 5 min) and incubated for 15 min with 4 0 ,6 0 diamino-2-phenylindole (DAPI) solution (Merck, Italy; dilution 1 : 2500 in deionized water). After being washed twice, images were acquired with a fluorescence microscope (Leica DM IRBE, Leica Microsystems Srl, Italy).

RNA extraction and Real Time PCR
Total RNA was isolated using the ReliaPrep™ RNA Miniprep System (Promega), following the manufacturer's instructions. Then, 1 mg of RNA was reverse transcribed to cDNA with the iScript cDNA synthesis kit (Bio-Rad), according to the manufacturer's instructions. Real time PCR was performed with 10 ng of cDNA template, 0.2 mm primers, and 1× GoTaq® qPCR Master Mix (Promega) in 20 ml of final volume, using a StepOnePlus® real time PCR system (Applied Biosystem). The amplification protocol was: 95°C for 2 min, 40 cycles of 5 s each at 95°C, 30 s at 57°C and 30 s at 72°C, and a final stage at 72°C for 2 min. Relative quantification of target genes was calculated by the equation 2 −ΔΔCt using two housekeeper genes (S14 and UBC). The primer sequences are reported in Table 1.

Western blot
For protein expression analysis, cardiac fibroblasts were lysed with RIPA buffer (1% Nonidet P-40 in 50 mm Tris-HCl, pH 7.5, 150 mm NaCl, 0.1% sodium deoxycholate, 1% protease inhibitor cocktails), incubated in ice for 30 min, and then centrifuged at 13 000×g for 15 min at 4°C. The supernatant was collected, and the total amount of proteins was determined with BCA assay (Pierce), following the manufacturer's instructions. Proteins (20 mg) were resolved on a 10% SDS-PAGE gel and subsequently transferred onto nitrocellulose membranes by electroblotting. The total amount of transferred proteins, used for the normalization of detected proteins, was determined with the REVERT Total Protein Stain kit (LI-COR Biotechnology), following manufacturer's instructions. Membranes were incubated with blocking buffer (TBS: 10 mm Tris-HCl, pH 7.4, 150 mm NaCl, 0.1% (v/v) Tween 20 containing 5% (w/v) dried milk or 5% (w/v) bovine serum albumin) for 1 h at RT, and then the primary antibodies were added and incubated overnight at 4°C in the proper blocking The membranes were washed three times with TBS-Tween 20 for 10 min and then incubated for 2 h with the appropriate secondary antibody. The secondary antibodies used were: anti-mouse HRP conjugated (Amersham), anti-rabbit HRP conjugated (Amersham), IRDye 800CW anti-mouse (Licor), IRDye 800CW antirabbit (Licor), IRDye 680CW anti-mouse (Licor), and IRDye 680CW anti-rabbit (Licor). After three washes with TBS-Tween 20, proteins were detected with an ECL detection kit (Cyanagen) or with infrared acquisition at the proper wavelength with the LI-COR Odyssey Infrared Imaging System (LI-COR Biotechnology).

Sialidase activity assay
Cells were collected by scraping and resuspended in PBS containing protease inhibitor and phosphatase inhibitor cocktails (Merck). Pulse sonication was used to lyse cells (10 pulses of 0.5 s in ice). The lysate was centrifuged at 800×g for 10 min at 4°C, and the membrane fraction was then separated by centrifugation at 200 000×g for 20 min at 4°C with a TLC100 Ultracentrifuge (Beckman Coulter). Total protein content was determined with the BCA protein assay kit (Pierce), following the manufacturer's instructions. The sialidase activity present in the membrane fractions was assayed using 4-MU-NeuAc at pH 3.8 according to a well-established protocol [35]. One milliunit of sialidase activity is defined as the amount of enzyme liberating 1 nmol of product (4-MU) per min.

Treatment of cell cultures with [3-3 H]sphingosine
The determination of the GM3 content in scramble, Neu3 overexpressing and shGM3 cells was performed by radioactive metabolic labeling, as previously described [38]. [3-3 H]sphingosine (PerkinElmer Life Sciences) was dissolved in methanol, transferred into a sterile glass tube, and then dried under a nitrogen stream. The residue was dissolved in growth medium to obtain a final sphingosine concentration of 30 nM (corresponding to 0.4 mCi/100 mm dish). An amount of 1 × 10 6 cells were incubated in this medium for a 2 h pulse followed by a 24 h chase, a condition warranting a steady-state metabolic condition. At the end of the 24 h chase, cells were harvested and lyophilized.

Lipid extraction and analyses
Total lipids from lyophilized cells were extracted twice with chloroform/methanol 2 : 1 (v/v) and with chloroform/methanol/water 20 : 10 :

GM3 silencing
Specific siRNA duplexes targeting GM3 synthase, siRNA transfection reagents, and reduced-serum transfection medium were purchased from Santa Cruz Biotechnology. The day before transfection, 7 × 10 5 cardiac fibroblasts were seeded in each well of a 12-well cell culture plate in DMEM low glucose, containing 10% FBS without antibiotics and incubated for 24 h at 37°C and 5% CO 2 . The next day, transfection complexes were prepared using GM3 synthase siRNA, siRNA transfection reagent, and transfection medium, according to the manufacturer's protocol, and were added to each well. The final concentration of GM3 synthase siRNA duplexes used was 3 mg. A scrambled siRNA (Santa Cruz Biotechnology) was used as negative control.

Statistical analysis
The Student's t-test or the one-way ANOVA, followed by the Bonferroni's multiple comparison test, were used to determine significance using GraphPad Prism 7 software. P values of less than 0.05 were considered to be significant. All P values were calculated from data obtained from at least three independent experiments. All error bars represent the standard deviation of the mean.

Fibroblasts activation upon TGF-β treatment
Isolated cardiac fibroblasts were phenotypically characterized by flow cytometry, analyzing the expression of the fibroblasts markers, of mesenchymal markers, and of immune system cells markers, to assess the purity of the isolated population. As expected, the cells were highly positive for CD9, CD29, CD44, CD73, CD90, and CD105 [40], and with low or null positivity for CD34, CD45, CD106, CD117, and HLA-DR (Supplementary Figure S1) [41]. Then, fibroblasts activation was induced with TGF-β. The treatment caused a marked increase in α-SMA and collagen I at both mRNA ( Figure 1A,B) and protein levels ( Figure 1C,D). Moreover, cells showed a morphological alteration, characterized by the formation of actin stress fibers, a hallmark of myofibroblasts differentiation ( Figure 1E). Interestingly, TGF-β treatment induced an alteration in Neu3 genic expression and enzymatic activity. In particular, Neu3 mRNA expression and activity were significantly reduced by 25% ( Figure 2A) and 50% ( Figure 2B), respectively. Then, we analyzed the expression of Sp1 and Sp3, the two principal transcription factors that control Neu3 expression, and both resulted significantly reduced of 35% ( Figure 2C) and 25% ( Figure 2D), respectively.

Neu3 overexpression reduces fibroblasts activation
Cardiac fibroblasts were infected with a lentiviral vector containing the human Neu3 sialidase gene or with a lentiviral scramble vector. Then, the mRNA expression and the catalytic activity of Neu3-overexpressing cells (NEU3) were assessed and compared with the scramble cells (SCR). The results showed an increase in both the  Figure S2A,B). Notably, the overexpression of Neu3 caused the significant decrease in the GM3 levels (2-fold), as expected (Supplementary Figure S2C). These results confirmed the effective overexpression of the active form of Neu3. SCR and NEU3 Data are presented as mean ± SD of three independent experiments. Scale bar 100 μm. Statistical significance was determined by one-way ANOVA (P < 0.01), followed by Bonferroni's test for multiple comparison. (*) P < 0.05. cells were then treated with TGF-β to induce myofibroblasts differentiation, and the expression of fibrosis markers was evaluated. α-SMA and collagen I showed a significant increase in mRNA expression in both cell lines ( Figure 3A,B); however, the increase in both genes in NEU3 treated cells was significantly lower than in SCR cells ( Figure 3C,D). Similar results were obtained in the analysis of protein expression, where a significant increase in α-SMA was observed in both cell lines upon TGF-β treatment ( Figure 3E), while collagen I was significantly increased only in SCR cells ( Figure 3F). In addition, the proteins increase in NEU3 treated cells was significantly lower than in SCR cells ( Figure 3G,H). These results were also confirmed morphologically by immunofluorescence analysis of α-SMA: there was a high increase in α-SMA staining in SCR cells after fibroblasts activation, compared with NEU3 cells, in which, conversely, the increase in α-SMA was lower ( Figure 3I). Moreover, analysis of the collagen deposition in the extracellular matrix by Sirius Red staining ( Figure 4A) was significantly increase after TGF-β treatment in both cell lines ( Figure 4B), but the increase was lower in NEU3 cells, compared with SCR cells ( Figure 4C).

Neu3 overexpression down-regulated TGF-β pathway activation
The TGF-β pathway is the main pathway implicated in fibrosis onset and progression [18] and GM3 promotes fibrosis through the stabilization of TGF-β R1 [25]. Thus, the activation of the TGF-β pathway after fibrosis induction was evaluated in NEU3 and SCR cells. Results showed an increase in the ratio between phospho-TGF-β R1 and total TGF-β R1 ( Figure 5A) and between phospho-SMAD2 and total SMAD2 ( Figure 5C) only in SCR cells. This increase was significantly higher, as compared with NEU3 cells (Figure 5B, D). These results indicate an activation of the TGF-β pathway only in SCR cells and not in NEU3 cells. In addition, NEU3 cells showed a significant increase in SMAD7 protein expression, a well-known inhibitor of the TGF-β pathway ( Figure 5E,F).

GM3 synthase silencing reduced fibrosis induction
To test whether the observed effects were due to NEU3-induced GM3 depletion and the consequent block of TGF-β pathway activation, we tested whether we could mimic NEU3 overexpression effects by silencing the GM3 synthase to reduce GM3 cell content. To this purpose, cardiac fibroblasts were transfected with specific siRNA duplexes targeting GM3 synthase. Analysis of the GM3 synthase mRNA and protein expression showed a reduction in 70% and 40%, respectively (Supplementary Figure S3A,B); moreover, also the total content of GM3 ganglioside resulted decreased of 30% in shGM3 cells (Supplementary Figure S3C). Then, cells were treated with TGF-β to induce fibroblasts activation. The results of the mRNA and protein expression of the fibrosis markers were similar to those obtained in the NEU3 cells: in particular, α-SMA and collagen I were increased in both SCR and shGM3 cell lines ( Figure 6A,B), even if the increase was significantly lower in shGM3 cells ( Figure 6C,D). The same results were also obtained in protein expression of α-SMA and collagen I: a significant protein increase was observed in SCR cells ( Figure 6E,F), and the increase in shGM3 cells was significantly lower compared with SCR cells (Figure 6G,H). Moreover, immunofluorescence analysis of α-SMA expression showed a strong increase only in SCR cells ( Figure 6E), and also the quantification of ECM proteins

GM3 synthase silencing inhibited TGF-β pathway
To test whether GM3 synthase silencing had any effect on fibrosis induction, the activation of the pathway was analyzed. As expected, upon TGF-β treatment, activation of the pathway was observed in SCR cells, as confirmed by the significant increase in both the phospho-TGF-β R1/TGF-β R1 ( Figure 8A) and phospho-SMAD2/SMAD2 ( Figure 8C) ratio. On the other hand, upon fibrosis induction, in shGM3 cells these ratios remained similar to those of untreated cells. Thus, the relative increase in both the phospho-TGF-β R1/TGF-β R1 and phosphor-SMAD2/SMAD2 ratio was significantly higher in SCR cells, as compared with NEU3 cells (Figure 8B,D). Moreover, the expression of the TGF-β receptor type-1 (TGFBR1) inhibitor SMAD7 was significantly increased in shGM3 ( Figure 8E), and this increase was significantly higher, as compared with SCR ( Figure 8F).

Discussion
In this work, we report that sialidase Neu3 expression and activity are down-regulated during TGF-β-induced cardiac fibrosis. Interestingly, we found a decrease in the two main transcription factors responsible for Neu3 transcription regulation, Sp1 and Sp3 [35], after TGF-β treatment that could explain the mechanism underlying Neu3 decrease. The Neu3 reduction supported the hypothesis that an induced activation of the Neu3 could counteract this degenerative process. Indeed, overexpression of the enzyme significantly decreases the effects of TGF-β on cardiac fibroblasts, reducing their activation toward the myofibroblasts phenotype, as Neu3-overexpressing cells expressed lower levels of α-SMA and they deposited less collagen in the ECM. Encouraged by these results, we investigated the mechanism of Neu3 activity. Indeed, the TGF-β pathway is known to be the principal regulator of fibrosis, and the activation of the signaling cascade is determined by the phosphorylation of TGF-β R1 and R-Smads, with Smad2 playing a central role in the process. Interestingly, Neu3 overexpressing cells, when exposed to TGF-β, exhibited lower levels of both phosphorylated TGF-β R1 and Smad2, indicating a reduced activation of the pathway. In addition, in NEU3 cells, higher levels of Smad7, one of the inhibitory members of the Smad family that acts as a negative feedback inhibitor of the TGF-β pathway, competing with R-Smads for binding with TGF-β R1 could be observed [42]. Overall, these data support that Neu3 overexpression was able to reduce TGF-β pathway activation. In this context, it is known that ganglioside GM3 participates in TGF-β signaling through a direct interaction with TGF-β R1. In fact, GM3 regulates serine phosphorylation of TGF-β R1, TGF-β R2, and Smad2/3, and it is essential for the epithelial-to-mesenchymal transition in human lens epithelial cells [25]. Thus, we envisioned that NEU3 effects could be mediated by the enzyme's ability to regulate GM3 levels. Along with this line, we previously demonstrated the pivotal role of Neu3 in regulating the intracellular levels of GM3 [31,34,35], also in NEU3 cardiac fibroblasts [36], even by the enzyme trans-activity on the gangliosides of adjacent cells [33]. To test our hypothesis, we mimicked the effect of Neu3 overexpression by silencing GM3 synthase. Remarkably, the reduction in both the mRNA and protein expression of GM3 synthase caused similar effects on fibrosis inhibition as we observed for Neu3 overexpression. Thus, these results support the notion that Neu3 effects could be mediated by a reduction in cellular GM3 levels that, in turn, could reduce the stabilization and activity of TGF-β R1. Indeed, the involvement of sialidase Neu3 in the fibrotic process has also been described in the lungs. However, Chen and coworkers [43] observed a decrease in Neu3 degradation, accompanied by an increase in its translation within lung epithelial cells, suggesting that Neu3 could behave as an inducer of pulmonary fibrosis. While these results seem to be in contrast with our data, it has been demonstrated that the response to TGF-β is organ- [44] and even cell-specific [45]. In fact, it is known that cardiac and pulmonary fibrosis significantly differ in their etiology [46]. In particular, the major source of the pulmonary mediators for fibroblasts activation and differentiation are the lung epithelial cells [46], whereas, in the myocardium, the onset is given by inflammation, which, in turn, triggers the fibroblasts-myofibroblast conversion [47]. Indeed, based on previous reports, the molecular response to TGF-β treatment is likely to be different in cardiac fibroblasts and epithelial cells [45]. Nonetheless, in our work, we found that Neu3 overexpression or GM3 synthase silencing could completely block TGF-β-induced fibroblasts activation. This apparent limitation could turn out being of critical importance from a translational perspective. In fact, after tissue injury, an initial fibrotic response has been shown to be critical for avoiding cardiac wall rupture, eventually maintaining organ integrity. On the other hand, an uncontrolled fibrosis progression leads to a deep remodeling of the heart, including chamber dilatation, cardiomyocytes hypertrophy, an increased risk of arrhythmogenesis, and the development of congestive heart failure [48]. Thus, the ideal antifibrotic therapy should be able to fine-tune fibrosis progression, and it is usually started after an unexpected initial acute event. To date, no efficient antifibrotic therapies are available to the clinic, and heart transplantation cannot be considered a therapeutic option for the general population. While new regenerative strategies are emerging [49,50], the identification of Neu3 and GM3 as possible new targets for pharmacological treatment is of great value. To this end, this work supports that sialidase NEU3 and GM3 are new players in cardiac fibrosis, and the modulation of their content should be further assessed for a possible therapeutic application.

Competing Interests
The authors declare that there are no competing interest Q2 ¶ s associated with the manuscript.

Funding Q3 ¶
This study was partially supported by Ricerca Corrente funding from Italian Ministry of Health to IRCCS Policlinico San Donato.