Slug Is Increased in Vascular Remodeling and Induces a Smooth Muscle Cell Proliferative Phenotype

Objective Previous studies have confirmed Slug as a key player in regulating phenotypic changes in several cell models, however, its role in smooth muscle cells (SMC) has never been assessed. The purpose of this study was to evaluate the expression of Slug during the phenotypic switch of SMC in vitro and throughout the development of vascular remodeling. Methods and Results Slug expression was decreased during both cell-to-cell contact and TGFβ1 induced SMC differentiation. Tumor necrosis factor-α (TNFα), a known inductor of a proliferative/dedifferentiated SMC phenotype, induces the expression of Slug in SMC. Slug knockdown blocked TNFα-induced SMC phenotypic change and significantly reduced both SMC proliferation and migration, while its overexpression blocked the TGFβ1-induced SMC differentiation and induced proliferation and migration. Genome-wide transcriptomic analysis showed that in SMC, Slug knockdown induced changes mainly in genes related to proliferation and migration, indicating that Slug controls these processes in SMC. Notably, Slug expression was significantly up-regulated in lungs of mice using a model of pulmonary hypertension-related vascular remodeling. Highly remodeled human pulmonary arteries also showed an increase of Slug expression compared to less remodeled arteries. Conclusions Slug emerges as a key transcription factor driving SMC towards a proliferative phenotype. The increased Slug expression observed in vivo in highly remodeled arteries of mice and human suggests a role of Slug in the pathogenesis of pulmonary vascular diseases.

Differentiation of SMC is regulated by transcriptional regulators of the myocardin-related transcription factor family (MRTF), such as myocardin and myocardin-like proteins 1 and 2 (MLK1 and MLK2) [11]. Myocardin (myoCD) increases the expression of actin cytoskeletal proteins via serum response factor (SRF). Conversely, SMC phenotypic switch is mediated by both the loss of positive differentiation signals and by the induction of multiple complementary repressor pathways, such as Krüppel-like factor 4 (KLF4) and transcription factor Sp1 (SP1) [12]. Interestingly, increased MLK1 expression has been involved in the phenotypic transition of endothelial cells (EC) in an in vitro model of endothelial injury [12]; and, SP1 appears to be the main transcriptional regulator of endothelial to mesenchymal transition (EnMT) in a model of inflammatory bowel disease [13,14], suggesting that transitional changes in cell phenotype involved in different clinical settings may be regulated by similar molecular pathways.
Slug belongs to the Snail transcription factor family that is involved in several biological functions, including epithelial to mesenchymal transition (EMT), cell differentiation, cell motility, cell cycle regulation, and apoptosis. Slug participates directly in the dissociation of cell-to-cell contacts by repressing endothelial cadherin (VE-cadherin) gene expression, and indirectly, by increasing extracellular matrix proteins [15,16]. Recently, the role of Slug in the control of different transcriptional programs of stem cell differentiation has been highlighted [17][18][19][20]; nevertheless its function in SMC has never been studied.
In the present study, we investigated the role of Slug in the phenotypic switch of SMC and its potential participation in the development of human pulmonary vascular remodeling. We showed that Slug induced SMC to undergo a proliferative phenotype by at least, modulating genes coding for cell proliferation and cell migration related pathways. Interestingly, we found increased levels of Slug, but not of Snail, in a mouse model of PH-related vascular remodeling and a positive correlation of Slug expression with the degree of both lung obstruction and arterial wall thickness. In addition, we observed Slug up-regulation in human pulmonary arteries with high degree of vascular remodeling. To our knowledge, this is the first time that Slug has been related to SMC proliferation and to vascular remodeling.
Briefly, cells were fixed using formaldehyde solution and incubated with X-gal. Pictures were taken after an ON incubation at pH 6. Blue senescent cells were counted and expressed as a percentage of the total number of nuclei DAPI-positive cells.

RNA Isolation and Real Time PCR
Total RNA was isolated using TRIzol 1 Reagent (Invitrogen) according to the manufacturer's instructions. Random-primed cDNA synthesis was performed at 37°C with 1 μg of RNA using the high capacity cDNA Kit (Applied Biosystems, Foster City, CA). Gene expression was measured by RT-PCR in a Chromo 4 Real Time PCR detector (Bio-Rad, Hercules, CA) and ABI fast 7900 HT (Applied Biosystems) using the sensiMix dt kit (Quantace, San Mateo, CA,) based on the DNA double-strand-specific SYBR green I dye and Taqman probes (Applied Biosystems) for detection. The results were normalized to GAPDH and β-actin expression levels and relative gene expression was analyzed by the 2-ddct method. The primers used and their sequences are listed in S1 Table. Western Blotting SMC were washed with PBS and then lysated with RIPA buffer (50 mM TRIS-Cl, 150 mM NaCl, 1 mM EDTA, 10% NP-40, 0.10% deoxycholic acid) containing protease inhibitors (Sigma-Aldrich). The lysate was centrifuged at 14.000g for 15 min at 4°C to pellet debris and the protein-rich supernatant was stored at -20°C. Total protein concentration was measured with Bradford (Bio-Rad Laboratories, Hercules, CA). Mixed samples of three experiments per line containing 60 μg of protein were run in a SDS-PAGE on 5-12% Bis-Tris gel (Biorad) before transferring to a PVDF membrane. Blocking was done with 5% BSA (Sigma) and samples were incubated ON with primary antibodies. Primary antibodies against Slug (Cell Signalling, dilution 1/1000), and β-actin (Cell Signalling, dilution 1/1000) were revealed with HRPlabeled secondary antibody (Upstate, Charlottesville, VA). Blots were exposed with enhanced chemiluminescence (ECL) (Pierce, Thermo Scientific) and visualized in a LAS 4000 lumi-Imager (Bio-Rad). Membrane densitometry was analyzed with ImageJ software (Public domain). Blots were stripped using stripping buffer (50 mM Tris, 20% SDS and 0.7% ß-mercaptoethanol) and reprobed.
Immunodetection SMC differentiation was assessed by immunofluorescence using antibodies against α-SMA (1/750) and calponin (1/75) (DAKO Cytomation, Carpinteria, CA) and SM-MHC (1/250) (Abcam, Cambridge, UK). An antibody against antigen Ki-67 (1/50) (Novocastra 1 , Newcastle, UK) was used to measure cell proliferation. Briefly, cells were washed twice with PBS and fixed with 4% paraformaldehyde for 30 min. After permeabilization with PBS-0.1% triton, cells were washed and incubated with the appropriate antibody ON at 4°C. All primary antibodies were revealed with a secondary antibody conjugated with fluorescein during 90 min (Jackson Immuno Research, West Grove, PA) at room temperature. Nuclei were stained with DAPI. Immunofluorescence images were quantified by counting the number of immunoreactive cells with respect to the number of nuclei. fresh growth medium. Pictures were taken at baseline and every 12 h. The healing area was analyzed with Image-Pro Plus software (Media Cybernetics, Inc.)

Knockdown and overexpression experiments
To study Slug overexpression, 1 x 10 6 cells were transfected with XL6 plasmid and XL6-Slug using the Amaxa Biosystem (Lonza) following manufacturer's guidelines. One day after electroporation, cells were cultured with starved medium ON and stimulated with 10 ng/ml of TGFβ1 (Acris) for 24 h.
Slug knockdown was accomplished with 10nM of siPool against Slug (SiTool Biotech GmBH, Martinsried), which allows the use of low concentrations of siRNA minimizing off-targets effects [23]. A scrambled sequence was used as control. Both siPools were transfected using Lipofectamine RNAimax (Invitrogen).

Transcriptome-wide gene expression analysis
SMC were transfected with 10 nM siSlug and mock-transfected in two biological replicates. RNA was isolated 48 h after transfection and further processed for transcriptome-wide expression profiles using the Human Gene 2.1 ST array platform (Affymetrix, Santa Clara, CA). Raw probe set intensities were processed and summarized using the robust multi-chip analysis (RMA) algorithm [24] with a custom chip definition file (CDF) from Brain array [25]. Expression was assessed for each gene based on linear modeling of the log2-normalized gene intensities using the R package Linear Models for Microarray Data [26]. Per-gene log2-fold changes and Benjamini-Hochberg false discovery rate (FDR)-adjusted p-values were estimated for Slug knockdown versus control. Slug knockdown samples in our array-based gene expression analysis, determined 355 (1200) genes that were differentially expressed with adjusted p-values of less than 0.01 (0.05).

In vivo experiments
All procedures involving animals and their care were approved by the Ethics Committee of the University of Barcelona and the Institutional Committee of the University of Valladolid for Animal Care and Use. Furthermore, they were conducted according to institutional guidelines in compliance with national (Generalitat de Catalunya decree 214/1997, DOGC 2450) and international (Guide for the Care and Use of Laboratory Animals, National Institutes of Health, 85-23, 1985) laws and policies.
Our mouse model of pulmonary hypertension included 16 female mice (C57/bl6 mice around 22-25 g) that were divided in three groups. One group was maintained in normoxia and inoculated with vehicle (control, CTL; n = 6); the second group was exposed to chronic hypoxia using an initial ramp cycle to reach 10% O 2 , 5% CO 2 for 3 weeks (chronic hypoxia, CH; n = 5); and the third group was exposed to CH and injected weekly with the VEGFR inhibitor, Sugen 5416 (Tocris, R&D Systems, Minneapolis, MN) at 20 mg/Kg (sub/cut) using a 25 gauge needle (CH+SU5416; n = 5) for three weeks. At the end of exposure, right ventricular pressure (RVP) was determined under anesthesia by inserting a pressure transducer (Milla, Houston, TX) into the right ventricle via the jugular vein. Animals were then sacrificed and their lungs processed for histology. The number of intrapulmonary vessels with a diameter <50 μm showing positive immunostaining for α-SMA were counted and expressed as a percentage of the total number of small vessels. The ventricle hypertrophy (Fulton index) was evaluated as the ratio between the right ventricle (RV) and left ventricle plus the septum weight.

Pulmonary artery isolation
Segments of pulmonary artery were obtained from 19 surgical lung specimens that were resected for the treatment of localized lung carcinoma. The study was approved by the Ethics Committee of the Hospital Clinic, Barcelona, Spain. Arterial wall morphometry was analyzed in cross-sectional rings of pulmonary arteries after elastic orcein staining, as previously described [27]. Pulmonary arteries measuring approximately 2 cm long with an external diameter of 1-2 mm were dissected under microscope and cleaned of surrounding parenchyma and connective tissue. Segments were preserved in RNAlater 1 solution (Ambion, Grand Island, NY) and frozen at -20°C until RNA extraction using TRIzol 1 Reagent (Invitrogen, Grand island, NY). RNA quality was checked with the LabChip 1 Test kit using the Agilent 2100 bioanalyzer (Agilent Technologies, Santa Clara, CA).

Morphometry
The area of the intimal layer, expressed as a percentage of the cross sectional area of the pulmonary artery, was used to classify arteries into 3 groups by establishing the 33 and 66 percentiles as cut-off values. The different groups were identified as less remodeled (R1), mildly remodeled (R2) and highly remodeled (R3) pulmonary arteries.

Immunohistochemistry
Slug expression was measured by immunohistochemistry in serial sections of highly remodeled (n = 3) and non-remodeled pulmonary arteries (n = 5). After hydration of the samples, bovine serum Albumin was used to block non-specific antibody interactions. The antibodies against Slug (1/75) (Cell Signaling, Boston, MA,) were added to the samples for 1 h at room temperature. After two rounds of washing, the secondary antibody was added for 45 min and contrasted with hematoxylin stain at room temperature. Areas positive for Slug in the arterial section were quantified with Image Pro and normalized to the total vessel wall area.

Statistical analysis
All values are reported as mean ± SE. Determinations were performed in duplicate and at least 3 independent experiments were performed for each set of conditions. Two-group comparisons were analyzed using the two-tailed paired Student t-test for dependent samples or with the Mann-Whitney Rank Sum test for non-normally distributed data. Group comparisons were performed using one-way ANOVA or two-way ANOVA. Post hoc pairwise comparisons were made using the Student Newman-Keuls test for normally distributed variables or the Kruskal-Wallis and Dunn test for non-normally distributed variables. For all procedures, Pvalues lower than 0.05 were considered statistically significant.

SMC phenotypic change in vitro models
We analyzed the expression of contractile SMC markers in both cell-to-cell contact and TGFβ1 induced SMC differentiation and during TNFα-induced SMC dedifferentiation. Differentiated SMC phenotype induced by cell-to-cell contact was associated with higher expression of SMC contractile markers (myoCD, gata-6, sm22α, calponin and caldesmon) and decreased gene expression of the stem cell transcription factor KLF4 (Fig 1A). Fully differentiated phenotype at D6 of culture was also confirmed by the strong expression of α-SMA, calponin and SM-MHC fibers, determined by immunofluorescence (Fig 1A). Similar results were obtained when differentiation was induced by 48 h of TGFβ1 treatment (S1 Fig). Ki-67 expression  Fig 1B) and the number of cells in S Phase of the cell cycle, measured by flow cytometry ( Fig  1C) decreased concomitantly with SMC differentiation indicating that SMC differentiation was concomitant with the decrease of cell proliferation. No changes in cell senescence were observed in highly confluent cells compared with proliferative cells (S2 Fig), indicating that cell senescence is not induced by cell confluence. Dedifferentiation of fully differentiated SMC was achieved by 48 h of treatment with the inflammatory cytokine TNFα, as shown by the decrease of contractile genes and an increase of KLF4 expression (Fig 1D). α-SMA, calponin and SM-MHC fibers also decreased after 48 h of TNFα treatment (Fig 1D). Concordantly, increased proliferation was observed in TNFα-treated cells, as measured by both higher Ki-67 expression ( Fig 1E) and increased cells in the S phase ( Fig 1F).

Slug expression correlated with a dedifferentiated phenotype
We measured the expression of Slug and its related transcription factor Snail in these models. Expression of Slug and Snail were high in proliferative/dedifferentiated SMC (D0) and their expression decreased significantly to half in fully differentiated SMC (D6) in both cell-to-cell contact (Fig 2A) and TGFβ1-induced SMC differentiation (S1 Fig). Interestingly, induction of dedifferentiation by TNFα resulted in the increase of Slug expression but not of Snail expression ( Fig 2B).

Slug regulates proliferation and migration of SMC
In order to investigate whether Slug expression is associated with changes in the proliferative/ migrative phenotype of SMC, gain and loss of function of Slug were performed. Knockdown of Slug was achieved by using a SiPool against Slug, which resulted in the 90% of Slug knockdown (S3 Fig). Slug knockdown in highly proliferative cells resulted in a significant decrease in both Ki-67 expression ( Fig 3A) and cells in S phase (Fig 3B). By contrast, Slug overexpression increased the number of proliferating cells (Fig 3C). These results indicate that Slug expression sustain SMC proliferation. Migration, measured as the capacity of cells to resolve an in vitro wound was also measured (Scratch assay). Slug knockdown resulted in the reduction of migrating cells (Fig 3D), whereas Slug overexpression was associated with an increase in the migration rate of SMC (Fig 3E), indicating that Slug stimulates a migratory phenotype in SMC.

Slug regulates both TNF-α and TGFβ1-induced SMC phenotypic change
We reasoned that if Slug induces proliferation and migration in SMC, it could also regulate changes in SMC phenotypic switch. Slug knockdown prevented the TNFα-induced dedifferentiation of SMC denoted by the restored expression of myoCD, gata-6, calponin and KLF4 following TNFα treatment (Fig 4A). Transient overexpression of Slug disturbed the TGFβ1induced SMC differentiation, which resulted in a lower expression of both myoCD and calponin after 48 h of treatment and a decreased α-SMA and calponin fibers (Fig 4B and 4C).

Slug regulates genes related to cell migration and proliferation in SMC
Differential gene expression analyses using Affymetrix Human Gene 2.1 ST arrays were performed 48 h after Slug knockdown in SMC (Fig 5A and S1 Appendix). GO terms analysis of Biological Processes that are modified after Slug knockdown revealed that cell proliferation was one of the most altered pathways. In addition, GSEA analysis showed an enrichment of downregulated genes related to proliferation (Fig 5B and S3 Table). We validated the up-regulation of the tumor suppressor retinoic acid receptor responder protein 3 (RARRES3, also known as TIG3) after Slug knockdown ( Fig 5C). Furthermore, the decrease of two pro-proliferative related genes, Cyclin A2 (CCNA2) and Heparin-Binding Epidermal Growth Factor (HBEGF), after Slug inhibition are shown in Fig 5C. Increased levels of two known Slug targets, claudin1 (CLDNI) and keratin19 (KRT19) were also validated ( Fig 5C). RARRES3, CLDNI and KRT19 increased along differentiation, whereas CCNA2 and HBEGF decreased (S4 Fig). These results correlate well with our findings in siSlug-transfected cells.

Slug is increased in mice with severe pulmonary hypertension
Treatment of mice with chronic hypoxia (CH) in combination with the VEGFR inhibitor, SU5416 (CH+SU5416), reproduces the severe PAH observed in humans. As described previously [28], muscularization of vessels was increased in both CH and CH+SU5416, with respect to control mice (Fig 6A). RV hypertrophy was confirmed by the increase in the Fulton Index in both CH and CH+SU5416 (Fig 6B). Right ventricular pressure (RVP) was increased in animals exposed to CH or CH+SU5416 (Fig 6C). RT-PCR analysis in lung homogenates showed a moderate increase of Slug expression in CH and CH+SU5416 animals ( Fig 6D). There were no statistically significant changes in the gene expression of Snail in CH animals compared with control mice (Fig 6D). A positive correlation was found between Slug expression and both the number of vessels positive for α-SMA and the Fulton Index (Fig 6E and 6F). Accordingly, in vitro treatment of SMC for 24 h with CH or cigarette smoke extract (CSE), two well-known inducers of vascular remodeling, stimulated both the increase of Slug and SMC phenotypic switch (S5 Fig). Expression of Slug is enhanced in highly remodeled human pulmonary arteries Next, we also tested whether or not Slug expression was altered in human during vascular remodeling. Seven patients with COPD and eleven subjects with normal lung function (four non-smokers and seven smokers) were enrolled in our study (S4 Table). Patients with COPD had significantly lower forced expiratory volume in the first second (FEV 1 ), that is the most frequent index for assessing airway obstruction; lower FEV 1 /Forced vital capacity (FVC) ratio, which indicate increased airway resistance to expiratory flow; lower diffusing capacity for carbon monoxide (DLCO), which measures the ability of the lungs to transfer gas inhaled air to the red blood cells in pulmonary capillaries, and mild hypoxemia, than healthy controls. Groups formed according to their degree of remodeling were matched for subjects with COPD and subjects with normal lung function; and, there were no differences in respiratory variables between groups. RT-PCR analysis showed that expression of Slug, but not of Snail, was significantly upregulated in highly remodeled pulmonary arteries (R3) compared to less remodeled arteries (R1) (Fig 7A). Additionally, a positive correlation between Slug gene expression and  and totally muscularized (within 75-100% visible muscularization) intrapulmonary vessels with a diameter <50 μm analyzed in CTL: control group (n = 6), CH: animals exposed to chronic hypoxia (n = 5), CH+SU5416: animals exposed to chronic hypoxia plus Sugen 5416 (n = 5) show positive immunostaining for α-SMA in CH and CH+SU5416 animals. *p<0.05 by one way ANOVA. B-C, Fulton index (B) and Right ventricular pressure (RVP) (C) are increased in CH and CHSU5416. D, RT-PCR in lung homogenates showed increased Slug, but not Snail expression, in the group exposed to CH and CHSU5416 analyzed by one-way ANOVA. E, Correlation between Slug expression (1/dCt) with both the number of α-SMA positive vessels and F, the Fulton index (note that the lower the 1/dCt, the higher the Slug expression level). *p < 0.05 by Spearman analysis.  Analysis of Slug expression in human pulmonary arteries. A, Samples were classified according to the degree of vascular remodeling: low (R1), mild (R2) and high (R3). Slug but not Snail expression was significantly increased in R3 arteries compared with R1. *p<0.05 by one way ANOVA. B, Slug expression (1/ dCt) displays a positive correlation with the intimal enlargement of pulmonary arteries measured by % of intimal thickness of the pulmonary artery (r = 0.44, p<0.05 by Pearson test). Note that the lower the 1/dCt, the higher the expression level. C, Representative images of Slug immunostaining; Slug expression is significantly increased in dedifferentiated intimal pulmonary SMC and EC of highly remodeled pulmonary arteries (right image) compared with less remodeled (left image). Negative control is shown in the first picture. Slug positive areas were quantified in highly remodeled (n = 3) and less remodeled (n = 4) arteries and normalized by the total wall area. Highly remodeled pulmonary arteries display a significant increase in Slug positive cells compared with less remodeled arteries. *p<0.05 by t-test. D, Slug (red), α-SMA (green) and merged (yellow) immunofluorescence images. Slug expression is predominantly found in the intima layer of remodeled pulmonary arteries.
doi:10.1371/journal.pone.0159460.g007 the thickness of the intima layer was found (Fig 7B). Slug localization in cross-sectional artery rings was assessed by immunohistochemistry and co-immunofluorescence with α-SMA antibody. Slug was barely detected in the intimal layer of less remodeled arteries (Fig 7C). In contrast, highly remodeled arteries showed strong intensity in the intima and media layer (Fig 7D).

Discussion
Although many reports have described key factors involved in the regulation of the SMC phenotypic switch [3], the detailed molecular mechanisms driving this process are not yet fully understood. Furthermore, many of the described players in vitro have not been found to be associated with vascular disease. In this study, we show that the EMT-related transcription factor Slug is highly expressed in proliferative SMC, and its expression decreases significantly as maturity increases. Diminished Slug expression, both during differentiation and after knocking down its expression, correlated with a reduced proliferation, therefore, suggesting that Slug regulates the expression of genes related to these pathways. Our genome-wide transcriptome study revealed that Slug modulates the expression of many genes that regulate proliferation, which is consistent with cell cycle assays. Specifically, downregulation of Slug in SMC was related with a decreased expression of both HBEGF and CCNA2, whereas the genes CLDNI, KRT19 and RARRES3 increased their expression. HBEGF is a stimulator of SMC proliferation and it is known to play an important role in neointimal formation of a rat model [29]. The CCNA2 gene encodes for cyclin-A2, a protein involved in cell cycle progression. Nevertheless Slug is a transcriptional repressor. And, the expression of both the CCNA2 and HBEGF genes decreased during its knockdown, suggesting that they are indirect targets of Slug. Downregulation of Slug in SMC was related with a greater expression of two known Slug target genes, CLDNI and KRT19. These genes are involved in adhesion/migration pathways [30,31], which may explain the increased rate of migration observed following Slug overexpression. Interestingly, both genes are described as specific epithelial genes [30,31]. However, we observed expression of these genes in differentiated SMC cells and in Slug-knocked down SMC, indicating that they also play a role in this cell type. Expression of KRT19 has been also previously described in vascular smooth muscle and skeletal muscle [32,33] RARRES3 is a tumor suppressor [34] gene that maintains cell adhesion and decrease metastasis [35]. Concordantly, its expression was increased after Slug knockdown. Overall, these results indicate the important physiological role of Slug in cell proliferation and migration in SMC.
Our study revealed that Slug induces a proliferative/dedifferentiated phenotype in SMC. This was demonstrated in the Slug overexpression experiments. In these assays, Slug promoted an increased rate of proliferation and migration and blocked the TGFβ1-induced SMC differentiation. These results agreed with a number of previous works demonstrating the key role of Slug in regulating cell dedifferentiation, proliferation and migration. Slug upregulation was found to confer a stem cell-like phenotype in a breast cancer model and in epithelial corneal cells [36,37]. Recently, it was also shown that Slug blocked differentiation of both striated muscle cells and epidermal cells [17,18]. Slug is a zinc finger transcription factor that suppresses gene expression through binding to E-boxes in the promoter of its target genes [16,38]. In striaded muscle cells, Slug represses differentiation by competing with the bHLH transcription factor MyoD, which is the main transcription factor driving differentiation of these cells, for binding to the E-boxes of specific muscle genes [18]. Many smooth muscle-specific genes, including α-SMA, sm22α and SM-MHC, contain E-boxes in their promoters and are activated by class I bHLH transcription factors [39]. Also, twist1, a known inductor of EMT, has also been shown to bind to E-boxes in the promoter region of both sm22α [40] and α-SMA, and repress them in vitro and in vivo [40]. In our work, we observed a lack of activation of sm22α and α-SMA, following Slug knockdown in proliferative cells (S1 Appendix), demonstrating that Slug is not directly repressing them at this stage of differentiation. However, it cannot be discarded that Slug displaces bHLH transcriptional activators once these genes are activated in differentiated cells.
TNFα treatment to induce a phenotypic switch from fully differentiated SMC was used in this study. Elevated levels of TNFα, or systemic inflammation, have been associated with blood vessel remodeling [41,42]. Specifically, TNFα is thought to be involved in several chronic hypoxia-associated lung diseases like COPD [43,44]. In our model, TNFα induced Slug but not Snail up-regulation, and induced contractile protein downregulation in differentiated cells with a concomitant increase of a migrative/proliferative phenotype. These results correlate well with previous work where TNFα inhibited the contractile phenotype of cerebral vascular SMC, inducing pro-inflammatory/matrix-remodeling genes [45], and stimulated Slug expression in breast cancer cells [46]. We observed that preventing the upregulation of Slug by transfection of the siSlug, following TNFα treatment, blocked the inflammatory-induced SMC phenotypic switch. Interestingly, Slug knockdown was able to prevent the TNFα-induced up-regulation of KLF4, which is a known inductor of SMC dedifferentiation [47]. We also observed increased Slug, but not Snail expression, in lungs of mice during vascular remodeling and in highly remodeled human pulmonary artery samples. This interesting asymmetry observed in the behavior of Slug and Snail has been previously described in several studies. Shirley and colleagues described that the two factors are not induced by identical stimuli [48]. In addition, Slug, but not Snail mRNA was elevated during wound reepithelialization in vitro and, ex vivo and in vivo in mice [49]. Moreover, to induce EMT, Slug and Snail showed overlapping, as well as a range of other functions. Slug seems to act early to trigger EMT, whereas Snail acts later to complete the process [50,51]. Remarkably, Snail knockout mice died at gastrulation [52], while Slug knockout mice were viable [53]. Altogether, these studies clearly show that Slug and Snail are not functionally equivalent, although they share certain targets in cell differentiation processes. Interestingly, a significant number of Slug overexpressing mice died of cardiac hypertrophy and cardiac failure exhibiting the key role of this factor in the cardiovascular system homeostasis. In correlation with these observations, using a murine model of severe pulmonary hypertension induced by CH, we found greater expression of Slug in lung homogenates of CHexposed animals, as well as a correlation of Slug with the severity of vessel remodeling. These findings support a stimulating role of Slug in hypoxia-induced SMC proliferation. In correlation, in vitro treatment of SMC with CH or CSE, both of which are reported to regulate cell proliferation during vascular remodeling [54,55], induced the expression of Slug concomitantly with cell differentiation defects. Interestingly, we also found a correlation between the intima thickness of human pulmonary arteries and Slug expression. A recent report from Owens and colleagues, using a cell-tracing system, showed that more than 80% of medial SMC during intima hyperplasia in mice undergo phenotypic switch [47]. Also, EnMT, a process in which both Slug and Snail are involved, was also observed in vivo during the PH-related vascular remodeling (Fig 8A) [56]. In this work, we documented that highly remodeled arteries showed a strong intensity of Slug, but not of Snail, in the intima and media layer, indicating that a greater number of SMC-like cells in the intima could arise from the media through phenotypic switch of SMC, likely driven by Slug (Fig 8A).
In summary, Slug is revealed as a key transcription factor driving a proliferative/migratory phenotype of SMC. Our results indicate that in an inflammatory environment, the expression of Slug is induced in SMC, which in turn, stimulates a SMC proliferative/dedifferentiated phenotype, at least through modulating proliferation and migration genes (Fig 8B). To the best of our knowledge, this is the first time that Slug has been studied during the phenotypic switch of SMC. The increased expression of Slug observed in remodeled human pulmonary arteries, suggests that this transcription factor has an important role in the pathogenesis of pulmonary vascular impairment that is promoted by an inflammatory and/or hypoxic environment. Accordingly, therapies targeting Slug could be of potential interest in the treatment of pulmonary vascular disorders.
Supporting Information S1 Appendix. Summary Table of  CArG boxes, while bHLH transcription factors, like USF bind to E-boxes to activate and maintain expression of SMC specific markers. In this stage, Slug is minimally expressed. In consequence, expression of its known targets, like CDN1 and KRT19, as well as the tumor suppressor RARRES3, are high and migration is repressed. Expression of the cell cycle related genes, CCNA2 and HBEGF, are highly expressed and the rate of proliferation is very low. Under specific stimuli, such as inflammation and/or hypoxia, the expression of Slug is triggered. Expression of its target genes CDN1 and KRT19 are blocked and cell migration is activated. Moreover, expression of CCNA2 and HBEGF decrease and cells become proliferative. In this condition, the expression of the stem cell factor KLF4 is upregulated. Expression of MYCD and its binding to CArG boxes is also repressed resulting in decreased expression of SMC specific markers. MYCD, myocardin; SRF, serum response factor; MRTF, myocardin-related transcription factors; KLF4, krüppel like factor 4; CDN1, claudin1; KRT19, keratin 19; RARRES3, retinoic acid receptor related gene 3; CCNA2, cyclinA2; HBEGF, Heparin-Binding EGF-Like Growth Factor.