Abstract
Objective. High endothelin-1 (ET-1) and transforming growth factor-β (TGF-β) levels may induce in healthy endothelial cells (EC) an endothelial-to-mesenchymal transition (EndMT). The same cytokines are associated with fibrosis development in systemic sclerosis (SSc). Although EndMT has not been definitively shown in SSc, this process, potentially induced by a stimulatory loop involving these 2 cytokines, overexpressed in this disease might contribute to fibroblast accumulation in affected tissues. Macitentan (MAC), an ET-1 receptor antagonist interfering with this loop, might prevent EndMT and fibroblast accumulation.
Methods. EC, isolated from healthy controls (HC) and patients with SSc, were treated with ET-1 and TGF-β and successively analyzed for gene and protein expressions of endothelial and mesenchymal markers, and for Sma- and Mad-related (SMAD) phosphorylation. Further, in the supernatants, we evaluated ET-1 and TGF-β production by ELISA assay. In each assay we evaluated the ability of MAC to inhibit both the TGF-β and ET-1 effects.
Results. We showed that both TGF-β and ET-1 treatments induced an activation of the EndMT process in SSc-EC as reported in HC cells. The ELISA assays showed a mutual TGF-β and ET-1 induction in both SSc-EC and HC-EC. A statistically significant increase of SMAD phosphorylation after treatment was observed in SSc-EC. In each assay, MAC inhibited both TGF-β and ET-1 effects.
Conclusion. Our work is the first demonstration in literature that SSc-EC, under the synergistic effect of TGF-β and ET-1, may transdifferentiate toward myofibroblasts, thus contributing to fibroblast accumulation. MAC, interfering with this process in vitro, may offer a new potential therapeutic strategy against fibrosis.
Systemic sclerosis (SSc) is a chronic disease characterized by the activation of the immune system, fibroproliferative vasculopathy, and tissue fibrosis1. Vascular abnormalities are involved in many organ dysfunctions, including lung, heart, and kidney2, and probably drive the first symptom of the disease, the Raynaud phenomenon. Although different mediators have been identified as active players in the vascular remodeling in SSc, such as endothelin-1 (ET-1) and transforming growth factor-β (TGF-β)1,2,3, the mechanisms underlying SSc vasculopathy and how this damage may lead to fibrosis remain poorly understood.
In this setting, myofibroblasts are considered the effector cell in the fibrotic process. The differentiation of quiescent fibroblasts toward cells displaying a proliferating, matrix-producing, contractile phenotype has been shown to be a basic step in this process4. Further, the use of in vivo animal models in which fibrillar collagen-producing cells have been tracked, helped us to understand that both pericytes and resident fibroblasts are a source of activated myofibroblasts as shown during the evolution of chronic kidney fibrotic diseases5,6,7. Several reports8,9,10 showed that myofibroblasts may also originate through the mesenchymal transition of endothelial cells (EC), the so-called endothelial-to-mesenchymal transition (EndMT), first observed in studies on cardiac development11, and to date, emerging as a possible pathogenetic mechanism in different diseases, including diabetic nephropathy, cardiac fibrosis, intestinal fibrosis, pulmonary hypertension, and SSc12,13,14,15,16.
During EndMT, resident EC delaminate from the polarized cell layer and invade the underlying tissue. This EC “mesenchymal” phenotype is characterized by the loss of cell-cell junctions and EC markers, such as Von Willebrand factor (vWF), CD31, and vascular endothelial-cadherin (VE-cadherin), as well as the acquisition of invasive properties associated with the gain of mesenchymal markers, such as α-smooth muscle actin (α-SMA), smooth muscle 22 (Sm22), and collagen1 (Col1A1)11,17,18,19. Despite evidence suggesting that EndMT is involved not only in pathological12,13,14,15,16,17,18,19 but also in physiological conditions20, the underlying molecular mechanisms involved in this process are largely unknown. Evidence has shown the crucial role of TGF-β signaling in the initiation of EndMT17. Of note, the same molecule is also considered a pivotal player in many fibrotic diseases, including SSc21,22,23. TGF-β exerts its profibrotic role by binding with specific receptors, and the signal is transduced to the nucleus by members of the Sma- and Mad-related (SMAD) family. It has been shown that TGF-β may induce phosphorylation of both SMAD1 and SMAD5, together with phosphorylation of SMAD2/3, in different cell lines such as EC, epithelial cells, fibroblasts, and cancer-derived cell lines24,25,26. The activation of intracellular transcription factors supported the production of other fibrotic molecules, such as ET-127. Further, TGF-β–mediated ET-1 release has been associated to the fibrotic response observed in scleroderma fibroblasts in the context of skin and lung fibrosis28.
ET-1, a 21–amino acid peptide, is known to be one of the most potent vasoconstrictors. In addition to its role in regulating vascular tone through its interaction with 2 specific receptors, endothelin receptor A (ETRA) and B (ETRB), this peptide further displays some fibrotic activity. In vitro studies showed that ET-1 promotes myofibroblast switch in fibroblasts, including the SSc fibroblasts19.
A better understanding of the mechanisms responsible for EndMT may be of primary importance in recognizing clinically useful biomarkers, predicting fibrotic remodeling, and/or developing effective antifibrotic therapies in different fibrotic conditions. Considering that EndMT may induce a profibrotic phenotype and contribute significantly to the vessel’s instability and capillary rarefaction during SSc, we investigated the ability of ET-1 in inducing EndMT in SSc-EC and the possible role of macitentan (MAC) in blocking this process. MAC is a novel ETRA/ETRB antagonist, showing in a longterm event-driven Phase III trial (SERAPHIN) the ability to reduce the risk of morbidity and mortality in patients with pulmonary arterial hypertension29.
To our knowledge, ours is the first report showing that SSc-EC may undergo EndMT and that ET-1 strongly modulates this process. Further, EndMT may be partially blocked by MAC. Our data suggest that this new ET-1 antagonist, interfering in vivo with this process, might offer a new therapeutic opportunity for fibrosis in SSc, a condition still lacking an appropriate therapy.
MATERIALS AND METHODS
EC isolation and culture
After obtaining approval from the San Salvatore University Hospital ethics committee and written informed consent from patients, microvascular EC were acquired from 10 patients with SSc with the diffuse cutaneous form of recent onset (disease duration < 3 yrs calculated since the first non-Raynaud symptom of SSc)30,31 by skin biopsies. Demographic and clinical characteristics of the patients are shown in Table 1.
Patients discontinued corticosteroids, oral vasodilators, intravenous prostanoids, or other potentially disease-modifying drugs at least 1 month before biopsies. None took immunosuppressants.
Ten frozen healthy control (HC) EC samples obtained from age-matched women donors (skin samples for research purposes) were used as controls.
Biopsy samples (1 × 0.5 cm) of the involved forearm skin (skin score 1/2 at the biopsy site) were washed with phosphate buffered saline (PBS; Life Technologies), and 4 explants were placed into a 50-ml tube containing 15 ml of trypsin (Sigma-Aldrich) and then to digest for 45 min at 37°C. Cells were cultured in EGM2-MV (Lonza) at 37°C in a humidified atmosphere of 5% CO2.
Before the cells reached confluence, after about 1 week, the heterogeneous pool of cells was exposed to a CD31-positive selection performed with the Dynabeads magnetic CD31 MicroBeads cell-sorting system (Invitrogen, Life Technologies). The beads rapidly targeted and partially coated the EC expressing the CD31 receptor.
After incubation, the cells were placed in a magnet (Dyna MPC-S; Invitrogen, Life Technologies) for 2 min, following the manufacturer’s recommended protocol for washings and final extraction. The CD31-negative cells were removed during the successive washings. The positive-selected cells were 99% EC with specific phenotype (CD-31, CD-34, CD-144; Figure 1A). The cells were used at third passages (P3).
EC treatment with TGF-β, ET-1, and MAC
To establish the optimal concentrations of TGF-β (R&D), ET-1 (Sigma-Aldrich), and MAC in our system, a dose/response curve was performed on α-SMA expression (data not shown) using P3 EC obtained from both 1 control and 1 patient.
Each experiment was performed in triplicate and the optimal stimulation dose for TGF-β was assessed to be 10 ng/ml, for ET-1, it was assessed to be 200 nM, and for MAC, it was 1 µm.
For EndMT gene and protein expressions, EC were treated in the following conditions: (1) untreated (UT) EC, (2) EC + TGF-β (10 ng/ml), (3) EC pretreated (1 h) with MAC (1 µm) before being treated with TGF-β, (4) EC + ET-1 (200 nM), and (5) EC pretreated (1 h) with MAC (1 µm) before being treated with ET-1. EC were cultured for 6 days in accordance with published studies19,32.
To assess SMAD 1/5 and SMAD 2/3 phosphorylation, EC were treated in the following conditions: (1) UT EC, (2) EC + TGF-β (10 ng/ml), (3) EC pretreated (1 h) with MAC (1 µm) before being treated with TGF-β, (4) EC + ET-1 (200 nM), and (5) EC pretreated (1 h) with MAC (1 µm) before being treated with ET-1. The experimental conditions were applied for 24 h.
Quantitative real-time (qRT)-PCR analysis
Total RNA was extracted from EC using NucleoSpin RNAXS (Macherey Nagel) according to manufacturer’s instructions and reverse transcribed into cDNA with the ThermoScript reverse transcription–PCR system (Invitrogen, Life Technologies).
The qRT-PCR was performed using SYBR green kits (Applied Biosystems). Results were analyzed after 45 cycles of amplification using the ABI 7500 Fast Real Time PCR System. Primers were designed on the basis of the reported sequences [Primer bank National Center for Biotechnology Information: β-actin: 5′-CCT GGC ACC CAG CAC AAT-3′ (forward) and 5′-AGT ACT CCG TGT GGA TCG GC-3′ (reverse); vWF: 5′-AGC CTT GTG AAA CTG AAG CAT-3′ (forward) and 5′-GGC CAT CCC AGT CCA TCT G-3′ (reverse); CD31: 5′-CCA AGG TGG GAT CGT GAG G-3′ (forward) and 5′-TCG GAA GGA TAA AAC GCG GTC-3′ (reverse); VE-cadherin: 5′-GTT CAC GCA TCG GTT GTT CAA-3′ (forward) and 5′-CGC TTC CAC CAC GAT CTC ATA-3′ (reverse); Sm22: 5′-CCG GTT AGG CCA AGG CTC-3′ (forward) and 5′-GCG GCT CAT GCC ATA GGA-3′ (reverse); α-SMA: 5′-CGG TGC TGT CTC TCT ATG CC-3′ (forward) and 5′-CGC TCA GTC AGG ATC TTCA-3′ (reverse); Col1A1: 5′-AGG GCC AAG ACG AAG ACA GT-3′ (forward) and 5′-AGA TCA CGT CAT CGC ACA ACA-3′ (reverse)]. Each gene was normalized to those for β-actin.
Western blot
To perform Western blot assays, EC cells were pelleted, washed twice with PBS, and lysed in sodium dodecyl sulfate (SDS) sample buffer. The protein concentration was calculated by Bradford protein assay reagent (Bio-Rad). There were 50 μg of proteins separated by SDS-polyacrylamide gel and transferred to nitrocellulose membranes. After 1 h at room temperature in blocking buffer [5% nonfat milk in Tris-buffered saline/1% tween 20 (TBS/T)], the membranes were washed 3 times for 5 min each in TBS/T and incubated overnight at 4°C with the primary antibodies: CD31, VE-cadherin (R&D Systems), Sm22 (Abcam), vWF, α-SMA, Col1A1 (Santa Cruz), Phospho-SMAD1/5, and Phospho-SMAD2/3 (Cell Signaling), diluted in 5% bovine serum albumin in TBS/T. Following 3 washes with TBS/T, horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology) diluted in blocking buffer was added for 30 min at room temperature and washed 3 times with TBS/T. The detection was done with enhanced chemiluminescence detection electrochemiluminescent reaction (Amersham Pharmacia Biotechnology). All the results were normalized to the levels of proteins of UT HC-EC and normalized to the actin signal (Sigma-Aldrich). Immunoreactive bands were quantified with densitometry using ImageJ software (National Institutes of Health).
ELISA
The concentrations of TGF-β and ET-1 released in EC supernatants were determined by ELISA using Quantikine Human Immunoassay kits (all by R&D Systems) according to the manufacturer’s protocol.
Statistical analysis
GraphPad Prism 5.0 software was used for statistical analyses. Results are expressed as median (range). Because of the nonparametric distribution of our data, the Mann-Whitney U test was used as appropriate for analyses. Statistical significance was expressed by a p value ≤ 0.05.
RESULTS
Effect of MAC on endothelial marker expression in SSc-EC
As shown in Figure 1 (B, C, D), after TGF-β or ET-1 treatment, the mRNA expression of endothelial markers markedly decreased in both HC-EC and SSc-EC when compared with UT cells. Of note, MAC significantly reverted both the TGF-β and the ET-1 effects on HC-EC and SSc-EC. As shown in Figure 1E, these results were confirmed at the protein level by Western blotting analyses.
Effect of MAC on mesenchymal markers expression in SSc-EC
In both HC-EC and SSc-EC, TGF-β or ET-1 treatment induced a significant increase of mRNA expression of the myofibroblast markers when compared with UT cells (Figure 2A, B, C). MAC reverted both the TGF-β and the ET-1 effects on HC-EC and SSc-EC, mirroring the results obtained in the endothelial markers analyses. The Western blot in Figure 2D showed that at protein levels, the results mirror the changes observed in gene expression levels.
The synergic ET-1/TGF-β production in SSc-EC
ET-1 production was assessed using a specific ELISA assay before and after cells treatment with TGF-β for 24 h. The results showed that ET-1 was significantly increased in UT SSc-EC when compared with UT HC-EC [UT HC-EC: 5.35 pg/ml (4.00–6.40) vs UT SSc-EC: 11.20 pg/ml (9.60–12.70), p = 0.0002]. Further, TGF-β significantly induced ET-1 production with significantly higher levels in SSc-EC when compared with HC-EC [HC-EC + TGF-β: 10.90 pg/ml (9.50–12.40) vs SSc-EC + TGF-β: 21.65 pg/ml (19.70–23.50), p = 0.0002; Figure 3A].
We further evaluated the TGF-β production after ET-1 stimulation. Already in UT condition, SSc-EC produced significantly higher levels of TGF-β when compared with HC cells [UT HC-EC: 30.90 pg/ml (28.20–35.20) vs UT SSc-EC: 62.85 pg/ml (60.90–66.10), p = 0.0002]. ET-1 significantly induced TGF-β production, and this increase was significantly higher in SSc-EC when compared with HC-EC [HC-EC + ET-1: 60.10 pg/ml (57.90–63.90) vs SSc-EC + ET-1: 92.75 pg/ml (88.90–95.70), p = 0.0002; Figure 3B].
SMAD phosphorylation is inhibited by MAC treatment in SSc-EC
The Western blot in Figure 3C shows that in UT SSc-EC, the levels of SMAD phosphorylation were higher than in HC-EC. After TGF-β treatment, we observed a significant increase of SMAD phosphorylation in both HC-EC and SSc-EC, the latter significantly higher than HC-EC. After ET-1 treatment, in both HC-EC and SSc-EC, the SMAD phosphorylation was significantly increased when compared with UT EC. Further, after ET-1 treatment, the levels of SMAD phosphorylation in SSc-EC were higher than in HCEC. MAC significantly blocked the ET-1 effect on SMAD activation in both HC-EC and SSc-EC. Further, we observed that MAC significantly inhibited the SMAD phosphorylation induced by TGF-β.
DISCUSSION
To our knowledge, our work is the first to demonstrate that ET-1 induces EndMT in SSc-EC and that this pathologic process may be inhibited by MAC, a new ET-1 dual receptors blocker. It is well known that ET-1 is strongly upregulated in patients with SSc and an endothelial dysfunction is involved in SSc pathogenesis. In addition to its well-known vasoconstrictive action, ET-1 displays a wide range of biological effects such as proliferation, fibrosis, and inflammation, and involving different cell types. ET-1 induces a profibrotic phenotype in fibroblasts through the increased expression of extracellular matrix proteins, such as Type I and Type III collagen and fibronectin, as well as decreasing the expression of matrix metalloproteinase 133,34. On the other hand, much evidence supports the hypothesis that ET-1 may induce the α-SMA expression in human EC, as well as an increased collagen production, thus modulating their transdifferentiation toward myofibroblasts8,9,10. The recruitment of myofibroblasts in affected tissues, associated with the persistence of their elevated biosynthetic functions, may be considered as pivotal determinants for the extent and the progression rate of the fibrosis in SSc10.
Available literature suggests that myofibroblasts may derive from several sources, including the expansion of tissue resident fibroblasts, the migration of bone marrow-derived circulating fibrocytes, pericytes, and the epithelial cells that underwent epithelial-mesenchymal transition5,7. A more recent study showed that EC, under specific stimuli, can also be considered a source of myofibroblasts undergoing EndMT, and this phenotypic transition could mainly be of interest in SSc pathogenesis by providing a cellular link between vasculopathy and fibrosis10.
We showed that SSc-EC acquired a myofibroblast-like phenotype after treatment with ET-1, as previously observed for normal EC after treatment with ET-1 and TGF-β9,27. Further, to assess the ability of SSc-EC to mirror the behavior of normal EC, we stimulated these cells with TGF-β, also showing that this cytokine may modulate EndMT in SSc-EC. In fact, SSc-EC displayed a reduction of endothelial markers (CD31, vWF), as well as an increase of profibrotic markers, such as stress fibers (Sm22, α-SMA) and collagen, after ET-1 and TGF-β stimulation. It has been shown that the endothelial damage in SSc skin is characterized by a progressive loss of the endothelial-specific marker VE-cadherin35, a strictly endothelial-specific adhesion molecule located at junctions between EC36. It has been recently shown that during EndMT, VE-cadherin expression is reduced in EC undergoing transdifferentiation37. Of note, in our experiments, we showed that after ET-1 and TGF-β stimulation, SSc-EC displayed a significant decrease of VE-cadherin expression, suggesting the induction of the EndMT program in our cells. This evidence allows us to speculate that this profibrotic switch may be considered a normal response of EC in any condition characterized by an overexpression of TGF-β and ET-1, such as the physiologic wound healing or the pathologic response to an unknown trigger, as occurs during SSc.
We recently reported the pathologic role that perivascular mesenchymal cells may play in the fibrotic evolution during SSc38,39,40 and the results of our present work suggest that in the process leading to SSc fibrosis, not only perivascular mesenchymal cells but also EC may contribute to myofibroblast generation through the EndMT program.
In this setting, considering the role of ET-1 in triggering EndMT41, ET-1 blockade may represent an important and still unexplored target to preserve EC integrity, thus decreasing the fibroblast accumulation in SSc.
In our experimental model, MAC treatment significantly reverted the ET-1 effect, as expected, but interestingly MAC was also able to inhibit the TGF-β–mediated EndMT on both HC-EC and SSc-EC. The inhibitory effect of MAC on the EndMT induced by TGF-β suggests that ET-1 may represent the ultimate mediator of the TGF-β actions. In line with this evidence, it has been reported42,43 that in both EC and fibroblast, TGF-β induces the ET-1 gene promoter activity by SMAD activation. To clarify this aspect, we further assessed the ET-1 production after TGF-β stimulation by ELISA assays. Our results confirmed that TGF-β stimulation increased the ET-1 production by EC. Of note, SSc-EC displayed higher ET-1 production, already at basal levels, when compared with HC-EC. Further, we observed that ET-1 stimulation was able to increase the TGF-β production in both HC-EC and SSc-EC, the latter showing significantly higher values of TGF-β before treatment. Under our experimental conditions, UT HC-EC and UT SSc-EC did not show any change in their phenotypic features, although SSc-EC constitutively produced higher levels of ET-1 and TGF-β than HC-EC. We showed that HC-EC and SSc-EC in vitro begin their morphological changes after 6 days of strong stimulation by ET-1 and TGF-β, a setting mirroring the cytokines milieu in which these cells may be exposed in vivo, considering the number of these molecules produced by activated fibroblasts and inflammatory-recruited cells.
An alternative hypothesis to explain the inhibitory effect of MAC on TGF-β–induced EndMT could be the cooperation between TGF-β receptors and ETR in inducing this program. It has been shown that G-protein–coupled receptors such as ET-1 receptors may cross over and interact with the pathways of serine/threonine kinase receptors, such as the TGF-β Type I receptor (TβRI). Therefore, agonists of ETR may also activate TβRI signaling and their downstream products, such as phospho Sma and Mad (pSMAD)44,45. In this setting, the effects of ET-1 may be due to crosstalk between these 2 receptors induced by the hijack of TβRI signaling machinery by ETR46. Our results showed that, similarly to TGF-β, ET-1 induced the phosphorylation of SMAD in HC-EC and SSc-EC, confirming the hypothesis of a possible TGF-β/ET-1 receptor cooperation. Of note, in UT SSc-EC, the levels of pSMAD were significantly increased when compared with UT HC-EC. We may speculate that in our experiments at basal condition, the TGF-β signaling pathway was already activated as a result of the overexpression of TGF-β and ET-1 during SSc, thus conditioning the higher levels of pSMAD observed in SSc-EC. In this scenario, MAC, interacting with the ETR, might also inhibit the TβRI transactivation and consequent SMAD phosphorylation induced after both TGF-β and ET-1 stimulation46. A further speculative and unexplored aspect may be the block of TβRI. However, to date, unlike ET-1, we still do not have any licensed drug able to interfere with TβRI47, and from a clinical point of view, ET-1 receptor antagonist now represents the only possibility we have to modulate this functional complex.
We showed for the first time, to our knowledge, the finding that ET-1 induces a phenotypic switch toward mesenchymal cells in SSc-EC, characterized by a downregulation of endothelial markers and an increased expression of profibrotic genes and proteins. Taken together, our results point out the possible contribution of damaged endothelia in the generation of profibrotic cells, and support the hypothesis of a pathologic link between vascular and fibrotic alterations in the pathogenesis of SSc.
Focusing on the involvement of TGF-β and ET-1 as early mediators of both vascular and fibrotic components of SSc, we suggest that these molecules may be considered key therapeutic targets in the early phases of the disease. MAC, the new dual ETR antagonist (as shown in our in vitro study), in inhibiting the EndMT, may offer new potential therapeutic strategies that target the TGF-β/ET-1 loop, and may prevent the early pathways leading to fibrosis in SSc.
Further studies are needed to translate these preclinical findings into the clinical setting. In fact, although many in vitro studies suggested the antifibrotic effect of another ET-1 receptor antagonist, bosentan42,48, no clinical benefits were observed when this drug was used in patients with SSc with lung fibrosis49. It must be taken into account that the failure of the clinical trials evaluating its efficacy on interstitial lung disease may be biased by the choice of a non-sensitive primary endpoint, as well as the heterogeneity of the enrolled patients and the lack of histological classifications. On this basis, any further study of the ET-1 receptor antagonist in patients with fibrosis might need more stringent inclusion criteria and perhaps primary and secondary endpoints that might be more responsive to change50.
Acknowledgment
The authors thank Federica Sensini for her technical assistance.
Footnotes
Supported by FIRA (Fondazione Italiana Ricerca per l’Artrite) 2009. G. Pecetti, S. Turricchia, and M. Iglarz are employees of Actelion Pharmaceuticals Ltd. P. Cipriani received a research grant from Actelion.
- Accepted for publication June 19, 2015.