The Compound LG283 Inhibits Bleomycin-induced Skin Fibrosis and Vascular Injury via Antagonizing TGF-β /Smad/Snail Mesenchymal Transition Pathways

Systemic sclerosis (SSc) is a collagen disease that exhibits intractable brosis and vascular injury of the skin and internal organs. Transforming growth factor-β (TGF-β)/Smad signaling plays a central role in extracellular matrix (ECM) production by myobroblasts. Myobroblasts may be derived from epithelial and endothelial precursor cells in addition to resident broblasts. Recently, our high-throughput in vitro screening discovered a small compound, LG283, that can disrupt the differentiation of dermal broblasts into myobroblasts. This compound was originally generated as a curcumin derivative. brosis and vascular injury through inhibition of TGF-β/Smad/Snail mesenchymal transition pathways and thus, may be a candidate therapeutic for treatment of SSc. Furthermore, the screening of EMT and/or EndoMT regulatory compounds may be an attractive approach for SSc therapy. stained for 20 min at 4 o C using indicated mAbs at predetermined optimal concentrations for 6-color immunouorescence analysis. Stained samples were assessed using a FACSCanto II (BD Biosciences) followed by data analysis using FlowJo software version 7 (Ashland). human umbilical vein endothelial cells; p-Smad3: phospho-Smad3; collagen bronectin-1; 4′,6-diamidino-2-phenylindole;


Introduction
Systemic sclerosis (SSc) is an autoimmune disease characterized by tissue brosis caused by excessive deposition of collagen and other extracellular matrix (ECM) components in the skin and visceral organs (1)(2)(3)(4). Activated broblasts and α-smooth muscle actin (α-SMA)-positive myo broblasts are mainly responsible for excessive synthesis and tissue deposition of ECM in SSc. Fibroblast activation and the phenotypic transition towards myo broblasts results from a complex series of events initiated by various pro-brotic molecules, including transforming growth factor-β (TGF-β), connective tissue growth factor (CTGF), platelet-derived growth factor (PDGF), and interleukin (IL)-4, IL-6, IL-13, IL-17A, and endothelin-1. Among them, TGF-β is likely the key molecule for functional activation of local broblasts and resultant tissue brosis in SSc (4,5).
Binding of activated TGF-β to its cell surface receptor triggers intracellular signal transduction of Smaddependent/-independent pathways. In the Smad-dependent pathway, activation of TGF-β receptor type I leads to phosphorylation of Smad2 and 3, allowing it to complex with Smad4 and translocate into the nucleus where it binds to Smad-binding element sequences of TGF-β responsive genes. Cofactors such as p300 are then recruited to the Smad-binding element-Smad complex, followed by transcriptional activation of the targeted genes. Excessive or dysregulated TGF-β/Smad signaling can result in ECM deposition and tissue brosis. Indeed, sustained signal activation has been detected in dermal broblasts of bleomycin-induced SSc mice (6) and inversely, blocking of the signaling cascade ameliorates skin brosis in experimental SSc models (7)(8)(9)(10)(11). However, no established TGF-β-targeted therapy has successfully been translated to SSc patients. TGF-β is well known to stimulate epithelial cells to undergo epithelial mesenchymal transition (EMT) in vitro (12,13). Recent reports of the close association between EMT and the establishment of SSc skin indicate; i) the epidermal basal layer of SSc skin exerts increased expression of vimentin and a Wnt gatekeeper secreted frizzled related protein 4, but inversely shows decreased expression of E-cadherin and caveolin-1 (14), ii) Twist-and Snail1-positive cells were found within eccrine glands of SSc skin (15), and iii) increased mRNA for SNAIL1, but not SNAIL2, was found in the SSc epidermis (16). Snail family zinc nger proteins are direct targets of the TGF-β cascade in epithelial cells and play a critical role in EMT during development, carcinogenesis, and tissue repair (17). Such an increase in gene expression caused by EMT-inducing transcription factors may thus promote some differentiation towards an active EMT process in the SSc epidermis.
Recently, EndoMT has also been indicated to be important in the pathogenesis of various brotic diseases and broproliferative vasculopathies including SSc (18)(19)(20). EndoMT can be induced mainly by TGF-β, although other factors, including endothelin-1, hypoxia, Wnt, Caveolin 1 and Notch, may also contribute to the process. Therefore, approaches to disrupt the process of EMT and/or EndoMT may be effective for the treatment of SSc.
Our high-throughput screening system identi ed LG283 from more than 1,200 compounds due to its ability to inhibit development of mesenchymal features in human dermal broblast cell lines. This compound was originally generated as a curcumin derivative and has been reported as a tau aggregation inhibitor by the name of PE859 (21). A previous paper demonstrated that this curcumin derivative is more effective than curcumin itself at inhibiting amyloidβ production (22). Therefore, this compound may be potentially useful for treatment of Alzheimer's disease. There are also many reports that demonstrate the anti-brotic activity of curcumin via inhibition of TGF-β/Smad signaling in various organs (23). Curcumin has been reported to inhibit TGF-β/Smad signaling via suppressing degradation of the TGF-induced factor, a negative regulator of TGF-β signaling, in SSc broblasts (24). However, the in vivo effects of curcumin or its derivatives on SSc have not been demonstrated.
In this study, we show that LG283 exhibits suppressive effects on brosis and vascular injury in both cultured human dermal broblasts and in a mouse model. Our ndings indicate that LG283 inhibits brosis and vascular injury mainly by antagonizing the TGF-β/Smad3/Snail pathway, which results in the augmentation of mesenchymal features of broblasts, epithelial, and endothelial cells.

Materials And Methods
LG283 LG283 was designed and synthesized as one of a series of curcumin derivatives with the original name of PE859 (3- Okuda and Sugimoto,et al (21).

Cell culture
Normal human dermal broblasts were purchased (Clontech) and were grown in Dulbecco's modi ed eagle medium (DMEM, Nacalai tesque) containing 10% fetal bovine serum (FBS), 100 U/ml penicillin and 100 μg/ml streptomycin (Nacalai tesque) at 37°C in a humidi ed 5% CO 2 atmosphere. When cells reached ~70% con uency they were starved in DMEM containing 0.1% FBS for 24 h and then pretreated with dimethyl sulfoxide (DMSO) as a control or indicated concentrations of DMSO-diluted LG283. Onehour after stimulation cells were stimulated with 10 ng/ml human recombinant (r) TGF-β1 (Peprotech) for use in experiments of immuno uorescence staining, real-time reverse transcription-polymerase chain reaction (RT-PCR), and western blot analysis. Fibroblasts between passages 3 and 5 were prepared for all experiments.

EMT
The A549 human non-small cell lung carcinoma cell line (American Type Culture Collection) was maintained in DMEM supplemented with 10% heat-inactivated FBS. Cells were seeded in 96-well plates at a density of 10,000 cells/well, 384-well plates at a density of 3,000 cells/well, and 3D-Nano-Culture Plates (SCIVAX Life Sciences). EMT was induced in DMEM containing 5% FBS with 5 ng/ml human rTGF-β2 (R&D Systems) for each indicated interval, with or without an incubation with LG283 (0.5μM) as described previously (25).

Animal studies
Healthy female C57BL/6 mice aged 8-10 weeks (CLEA Japan), not just siblings, were used for a bleomycin-induced skin brosis model (27). Bleomycin (1 mg/ml in saline) or 0.9% NaCl was injected subcutaneously into the shaved back of the mice (150 μl in each injection), concurrent with daily oral gavage of either LG283 (40 mg/kg or 80 mg/kg in sterilized olive oil) or olive oil alone for 4 weeks. The LG283 doses were optimized on the basis of sequential pilot experiments (data not shown). The drug was administered at the same time in all treatment groups for each independent experiment. All mice were housed in the same room of a speci c pathogen-free barrier facility and screened regularly for pathogens.

RT-PCR
Total RNA was isolated from the skin or cultured broblasts using RNeasy spin columns (Qiagen) and was digested with DNase I (Qiagen) to remove chromosomal DNA. Total RNA was reverse-transcribed to a complementary DNA with random hexamers (Takara Bio). Real-time RT-PCR was performed using the StepOnePlus Real-Time PCR system (Applied Biosystems). All data were normalized against GAPDH mRNA and are expressed as relative expression.

Western blot analysis
Total protein was extracted from human dermal broblasts or A549 human non-small cell lung carcinoma cell line using a 101 Bio Cytoplasmic & Nuclear Protein Extraction kit (Medibena). Samples of bleomycin-injected skin were homogenized in 600 μl of lysis buffer (10 mmoles/liter phosphate buffered saline, 0.1% SDS, 1% Nonidet P40, 5 mmoles/liter EDTA containing complete protease inhibitor mixture [Roche Diagnostics]) to extract proteins. Protein concentration was quantitated using a BCA protein assay kit (Takara Bio) on a spectrophotometer. An equal amount of protein was subjected to a standard SDS-PAGE and was transferred to a nitrocellulose membrane (Bio-Rad Laboratories Inc.). The blotted membrane was blocked for 30 min at room temperature with 5% skim milk/TBS, followed by incubation with anti-human antibodies to collagen type I alpha 2 chain (Col1A2, Abcam), bronectin-1 (FN-1, LS bio), phospho-Smad3 (p-Smad3, Cell Signaling Technology), SNAIL1 (Gene Tex), SNAIL2 (Invitrogen), ZEB1 (Abcam), ZEB2 (Gene Tex), Twitst 1 (Santa cruz biotechnology), GAPDH (Thermo Fisher Scienti c), TUBULIN (Bio-Rad Laboratories Inc.) or anti-mouse antibodies to Smad3 (Invitrogen) , p-Smad3 (Invitrogen), GAPDH (Sigma-Aldrich) overnight at 4 o C. After washing with TBS-T three times, the membrane was incubated for 1 h at room temperature with HRP-conjugated secondary antibody. The protein bands were visualized using Chemi-Lumi One Super solution (Nacalai tesque). All data were normalized against GAPDH or TUBULIN expression and are expressed as relative expression.

Histologic analysis
Para n-embedded mouse skin sections (6 μm in thickness) were subjected to hematoxylin-eosin and Masson's trichrome staining. For evaluation of skin brosis, dermal thickness was de ned computationally as the skin thickness from the top of the granular layers to the junction between the dermis and subcutaneous fat (28). Data were assessed in ve distinct elds under an equal magni cation (x 40) using a light microscope, and are expressed as mean ± SEM. Each section was examined independently by two investigators (T.C. and N.O.) in a blinded manner. Collagen deposition was quanti ed on Masson's trichrome-stained sections as the ratio of blue-stained area to total stained area using Adobe Photoshop Elements version 12.

Cytometric Bead Array
Concentrations of cytokines in skin were determined by using the Cytometric Bead Array Mouse In ammation Kit (BD Biosciences). Samples (40 mg) of bleomycin-injected skin were homogenized in 600 μl of lysis buffer (10 mmoles/liter phosphate buffered saline, 0.1% SDS, 1% Nonidet P40, 5 mmoles/liter EDTA containing complete protease inhibitor mixture [Roche Diagnostics]) to extract proteins. Homogenates were centrifuged at 15,000 revolutions per minute for 15 minutes at 4 °C to remove debris, and the supernatants were used for the measurement of cytokines.
Preparation of skin cell suspensions A 2×2.5-cm piece of depilated back skin was minced and digested in 7 ml of RPMI 1640 containing 10% FBS with 2 mg/ml crude collagenase (Sigma-Aldrich), 1.5 mg/ml hyaluronidase (Sigma-Aldrich), and 0.03 mg/ml DNase I (Roche Applied Science) at 37 o C for 90 min. Samples were passed through a 70-μm Falcon cell strainer (BD Biosciences) to obtain single-cell suspensions. After centrifugation at 1,500 rpm for 5 min, the cell pellet was resuspended in a 70% Percoll solution (GE Healthcare) and then overlaid with a 37% Percoll solution, followed by centrifugation at 1,800 rpm for 20 min. The cells were aspirated from the Percoll interface and passed through a 70-μm Falcon cell strainer. The harvested cells were washed with ice-cold PBS and were used for ow cytometric analysis.
Flow cytometry mAbs used were: alexa uor 488-conjugated anti-CD45, paci c blue-conjugated anti-CD11b, PerCPconjugated anti-Ly6C, and APC-conjugated anti-CD204 (R&D Systems). To distinguish dead cells from live cells, the Live/Dead Fixable Aqua Dead Cell Stain kit (Invitrogen) was used. The single-cell suspensions obtained above were stained for 20 min at 4 o C using indicated mAbs at predetermined optimal concentrations for 6-color immuno uorescence analysis. Stained samples were assessed using a FACSCanto II (BD Biosciences) followed by data analysis using FlowJo software version 7 (Ashland).

Statistical analysis
All data were analyzed using Graphpad Prism software version 7 and are expressed as mean ± SEM. The signi cance of differences between samples was determined with Student's 2-tailed t-test. P-values less than or equal to 0.05 were considered as statistically signi cant. Further details are available in the Supplementary Materials and Methods.

Results
LG283 inhibits TGF-β-induced expression of ECM in human dermal broblasts Excessive production of ECM by skin broblasts or myo broblasts contributes to skin brosis. Therefore, we examined the biological effects of LG283, a small compound that was detected as a candidate for anti brotic drug by our high-throughput in vitro screening, on ECM synthesis by cultured human normal skin broblasts. As assessed by real time-RT-PCR, baseline mRNA expression of COL1A2 and FN-1 was signi cantly increased by subsequent treatment with rTGF-β1 (Fig. 1a). In contrast, one-hour pretreatment with 4.5 μM of LG283 signi cantly suppressed the TGF-β1-dependent induction of both mRNAs to steadystate levels. A similar trend was observed for the protein expression of COL1A2 and FN-1, as assessed by western blotting (Fig. 1b). These ndings suggest that pretreatment with LG283 e ciently inhibits TGF-βinduced brogenic activity of skin broblasts.
LG283 inhibits the TGF-β-dependent increase of transcription factors responsible for the mesenchymal transition of human dermal broblasts The differentiation of broblasts into myo broblasts is critical for local ECM production and resultant brosis in the skin. Therefore, we investigated the expression of representative transcription factors responsible for the differentiation into myo broblasts in cultured, human normal skin broblasts (Fig.   1c). The mRNA expression of zinc-nger transcriptional regulators SNAIL1 and SNAIL2 was found to increase following treatment with rTGF-β1. However, pretreatment with LG283 signi cantly inhibited the TGF-β-dependent induction of both mRNAs. On the other hand, rTGF-β1 and/or LG283 did not alter the expression levels of ZEB1 and ZEB2, other zinc-nger transcriptional regulators associated with the transition into myo broblasts, suggesting LG283 speci cally inhibits the TGF-β-dependent mesenchymal transition cascade.
LG283 abrogates TGF-β-dependent phosphorylation of Smad3 in human dermal broblasts The TGF-β binding to its receptor induces the phosphorylation of Smad2/3 transcription factors upon canonical signaling. Phosphorylated Smad2/3 and cytoplasmic Smad4 intercommunicate to transfer the signal to the nucleus and result in the transcriptional gene regulation responsible for tissue brosis. We investigated the effects of LG283 on Smad phosphorylation in cultured human normal skin broblasts.
Immunocytochemical analysis revealed that treatment with rTGF-β1 increased cytoplasmic and nuclear staining for p-Smad3 (Fig. 1d). However, Smad3 phosphorylation was inhibited by pretreatment with LG283.
LG283 blocks TGF-β-induced EMT in cultured A549 lung epithelial cells We performed an EMT assay using the A549 human lung carcinoma epithelial cell line. When cultured on 2-D plates, A549 cells rapidly grew to a con uent epithelioid sheet-like appearance, which was not affected by the presence of LG283 (Fig. 2a, upper panels). Morphologically, the cells appeared round with loose clusters and sparse intercellular adhesions. Upon treatment with rTGF-β2 for 72 h, the cells changed to a broblastic spindle shape (Fig. 2a, left lower). However, simultaneous treatment with rTGF-β2 and LG283 somewhat negated the morphological change of A549 cells (Fig. 2a, right lower). On 3-D culture, A549 cells rapidly formed colonies of various sizes similarly in the presence and absence of LG283 (Fig. 2b, upper panels). Treatment with rTGF-β2 caused the cells to spread out to form colonies and decreased the amount of intercellular adherence and size of each colony (Fig. 2b, left lower), all of which were inhibited by simultaneous treatment with LG283 (Fig. 2b, right lower).
Next, we examined whether the LG283-dependent morphological stability is linked with epithelial and mesenchymal gene expression in 3-D cultured A549 cells. Treatment with rTGF-β2 markedly reduced the expression of E-cadherin mRNA, a representative epithelial marker, but inversely increased expression of mesenchymal markers such as FN-1, α-SMA, and CTGF at 48hr (Fig. 2c). The altered expression pattern of epithelial and mesenchymal markers was signi cantly repressed by simultaneous treatment with LG283 (Fig. 2c). Treatment with TGF-β2 increased the expression of SNAIL1 and SNAIL2 mRNA, but not that of ZEB1 and ZEB2 (Fig. 2c). The increased mRNA expression of SNAIL1 and 2 was suppressed by simultaneous treatment with LG283, although other transcription factors, ZEB1 and ZEB2, did not change in their mRNA expression (Fig. 2c). Similarly, protein levels of SNAIL1 and 2 were increased by TGF-β2.
However, the increase was reduced by simultaneous LG283 treatment (Fig. 2d). Protein levels of ZEB1 and 2 and TWIST1 were not signi cantly changed following treatment with TGF-β2 and/or LG283 at 96h (Fig. 2d). Western blotting exhibited the antagonizing effect of LG283 on TGF-β1-dependent p-Smad3 expression (Fig. 2e). Thus, LG283 signi cantly blocks TGF-β-induced EMT via speci c inhibition of Smad3 phosphorylation and subsequent Snail signaling in epithelial cells.
LG283 suppresses EndoMT in cultured HUVEC cells EndoMT may affect microvascular derangement and loss of functional endothelial cells leading to poor capillary bed formation, impaired angiogenesis, and chronic tissue ischemia in addition to tissue brosis. Indeed, endothelial dysfunction is considered a crucial factor for peripheral vessel remodeling in SSc (18, 29-31).
We used human endothelial HUVEC cells to examine the effects of LG283 in an EndoMT assay. Upon treatment with a cocktail containing rTGF-β2, TNF-α and IL-1β, the cells exhibited reduced expression of CD31 mRNA, a representative endothelial marker, and increased expression of FN-1 mRNA, a representative mesenchymal marker, by 24 h (Fig. 3a). However, simultaneous treatment with LG283 resolved the disparate mRNA expression patterns of decreased endothelial marker CD31 and increased mesenchymal marker FN-1 in response to the cytokine cocktail by 24 h and 48h, respectively (Fig. 3a). Next, the mRNA expression levels of EndoMT-associated transcription factors were investigated in HUVEC cells treated with the cytokine cocktail and/or LG283 for 48 hours. Expression levels of both SNAIL1 and SNAIL2 were remarkably increased following stimulation with the cytokine cocktail; however, this increase was signi cantly inhibited by simultaneous treatment with LG283 (Fig. 3b). On the other hand, the expression of ZEB1 and ZEB2 mRNAs were almost unchanged following treatment with the cytokine cocktail and/or LG283. Therefore, LG283 appears to suppress the effect of cytokines, including TGF-βinduced EndoMT, via speci c inhibition of Snail signaling in endothelial cells.
LG283 inhibits the development of bleomycin-induced skin brosis in mice Using a bleomycin-induced skin brosis mouse model, we examined the in vivo anti brotic effects of LG283. Subcutaneous bleomycin injection and oral LG283 were co-administrated daily for 4 weeks. No apparent side effects including the change of body weight and activity were observed in any mice (data not shown). Histologically, skin thickness was increased more than two-fold following bleomycin injection, which was signi cantly reduced by both doses (40 mg/kg and 80 mg/kg) of oral LG283 (Fig.  4a, upper columnsand b, left). Similarly, the Masson's trichrome-stained area was signi cantly reduced in bleomycin-injected skin sections from LG283 treated-mice, compared to those from mice treated with placebo (Fig. 4a, lower columns and b, right).
LG283 suppresses the reduction of capillary vessels in skin of bleomycin-treated mice To investigate the effect of LG283 on vascular injury, capillary vessels were stained with anti-CD31 antibody in bleomycin-injected skin on day 28. Subcutaneous bleomycin injection reduced the capillary vessels (Fig. 4c), similar to what is seen in the skin of SSc patients. However, simultaneous administration of oral LG283 signi cantly suppressed this decrease in capillary vessels in the skin (Fig.   4c). Thus, this suggests LG283 treatment is protective against destructive vascular injury during the process of skin brosis.
LG283 does not affect in ammatory cell in ltration during the early-stage of bleomycin-induced skin brosis Subcutaneous injection of bleomycin induces an early and transient in ammation mediated by locally in ltrating macrophages and other in ammatory cells (6). Local injection of bleomycin, but not control saline, induces increased in ltration of F4/80-positive macrophages into the dermal and subcutaneous tissues at day 7 (Fig. 4d). In addition, there was evident local in ltration of CD3-positive T cells in bleomycin-injected skin, but not in control skin (p<0.05; Fig. 4d). However, oral LG283 administration did not affect the in ltration of these cell subsets.
To further characterize the macrophage subset present in bleomycin-treated skin, we isolated CD11bpositve leukocytes from the lesional skin on day 21 and stained for monocyte/macrophage surface markers. As reported previously (32), proin ammatory macrophages (CD11b + Ly6C hi ) and pro brotic M2 macrophages (CD11b + CD204 + ) were both increased in bleomycin-injected skin. Oral LG283 did not signi cantly reduce the in ltration of macrophage subsets (Fig. 4e). Thus, LG283 does not appear to signi cantly affect the skin in ammation induced by bleomycin injection.
LG283 does not affect proin ammatory or pro brotic cytokine production in bleomycin-injected skin The process of early in ammation and subsequent brosis following subcutaneous bleomycin injection can be associated with increased production of various proin ammatory and pro brotic cytokines. In general, the concentrations of investigated cytokines, IL-2, IL-4, IL-6, IL-10, IL-17A, TNF-α and interferon (IFN) -γ, were increased in uid from bleomycin-injected skin at day 7 (Fig. 5a). Among these cytokines, the concentration of IL-10, a representative regulatory cytokine, in whole extracts from bleomycin-injected skin was signi cantly reduced by co-administration of oral LG283. However, oral LG283 treatment did not signi cantly change the concentration of proin ammatory cytokines, such as IL-2, IL-6, IL-17A, TNF-α and IFN-γ, or of a pro brotic cytokine, IL-4.
LG283 antagonizes the expression of phosphorylated Smad3 in bleomycin-injected skin TGF-β/Smad signaling has been considered to be essential for tissue brosis. Therefore, the effect of LG283 treatment on TGF-β/Smad signaling was evaluated in the bleomycin-injected skin at day 7. The expression of TGF-β1 mRNA in bleomycin-injected skin was not signi cantly affected by oral LG283 (Fig.  5b). Similarly, LG283 administration did not change the concentration of TGF-β1 protein in the bleomycininjected skin extraction uid (Fig. 5c). Expression levels of Smad3 protein were not affected by bleomycin and/or LG283 treatment (Fig. 5d). In contrast, expression of p-Smad3 protein was markedly increased in bleomycin-injected skin compared to controls, an effect that was signi cantly inhibited by LG283 treatment of mice (Fig. 5d). Thus, LG283 treatment speci cally inhibits the expression of the p-Smad3 in the skin of bleomycin-induced skin brosis model.
LG283 suppresses Snail expression in bleomycin-injected skin Since our in vitro ndings indicate that LG283 inhibits both TGF-β-induced EMT and EndoMT, we examined the in vivo expression of transcription factors associated with EMT and/or EndoMT. Similar to what was seen in vitro, expression of Snail1 and Snail2 mRNAs were signi cantly reduced in bleomycininjected skin following administration of oral LG283 on day 7 (Fig. 6a). However, expression of Zeb1, Zeb2, and Twist1 mRNAs were not signi cantly changed by oral LG283.
Consistent with these ndings, immunohistopathology showed that the expression of Snail1 and 2 were augmented in bleomycin-injected skin on day 7. However, oral LG283 inhibited the expression of both transcription factors in epidermal keratinocytes, follicular epithelial cells, and dermal broblasts (Fig. 6b).
Immuno uorescent staining revealed induction of Snail1 expression in CD31-positive endothelial cells and F4/80-positive macrophages in the dermis following bleomycin injection, an effect suppressed in mice treated with oral LG283 (Fig. 7). Thus, LG283 treatment speci cally inhibits expression of the Snail transcription factor in skin cells, including keratinocytes and endothelial cells, following bleomycin treatment.

Discussion
In this study, we investigated the inhibitory effects of the tau aggregation inhibitor, LG283, on skin brosis and vascular injury both in vitro and in vivo.
LG283 disrupted the TGF-β1-dependent increase of Smad3 phosphorylation, Snail1 and 2 expression, and development of major skin ECMs in cultured human skin broblasts. Moreover, LG283 ameliorated skin brosis and vascular injury in a mouse model induced by subcutaneous injection of bleomycin. The in vivo effects of LG283 were largely attributable to the suppression of p-Smad3 and overexpression of Snail1 and 2. However, this compound did not affect in ammatory cell in ltration or in ammatory cytokine concentration in the skin during the brogenic process. Our results illustrate the direct antagonistic effects of LG283 and its potential for therapeutic application for inhibition of mesenchymal differentiation and the brogenic response.
Dermal broblasts from SSc skin show constitutive phosphorylation and nuclear translocation of Smad2/3 with various levels of activated Smad signaling. Therefore, targeting the TGF-β/Smad signaling is an attractive strategy for the treatment of SSc. In the current study, we con rmed that LG283 inhibits the expression of COL1α and FN-1 in human dermal broblasts stimulated with TGF-β1. During the process, LG283 blocks Smad3 phosphorylation and expression of Snail 1 and 2, major downstream transcription factors speci c for the mesenchymal phenotype. Thus, LG283 shows anti-brotic activity via suppression of TGF-β/Smad/Snail signaling in dermal broblasts.
Accumulating evidence indicates that pro brotic myo broblasts may be derived from various precursors including pericytes, bone marrow-derived circulating cells, epithelial cells, endothelial cells, and adipocytes (33)(34)(35). In addition, EndoMT has been considered to be possibly important for the development of broproliferative and/or destructive vasculopathy and thereby may link the development of endothelial dysfunction/loss and skin brosis in SSc (18, 35). In this study, LG283 showed in vitro suppressive effects on EMT of human lung epithelial cells and EndoMT of human umbilical endothelial cells induced by TGF-β2 and a cytokine cocktail (TGF-β2, TNF-α, IL-1β), respectively. Additionally, LG283 inhibited the expression of p-Smad3 in lung epithelial cells stimulated with TGF-β2. Among various cytokine-transcriptional cascades involved in the process of EMT and EndoMT, Snail1 and 2 are dominant downstream transcription factors of TGF-β/Smad signaling and are likely to play central roles.
LG283 signi cantly inhibited the expression of both Snail 1 and Snail 2. Moreover, TGF-β1 stimulation did not increase the expression of Zeb 1 or 2, and the expression levels of these genes were not affected by LG283 treatment suggesting the mechanism is speci c. These ndings indicate that LG283 may antagonize Smad3 phosphorylation downstream of TGF-β as well as the subsequent upregulation of Snail1 and 2 in broblasts, epithelial cells, and endothelial cells during the brotic process.
Consistent with these in vitro ndings, oral LG283 administration was also effective against skin brosis and destructive vascular injury in a bleomycin-induced skin brosis mouse model. In this model, skin brosis is preceded by an increase of in ammatory cell in ltration and in ammatory cytokine expression(36). However, LG283 did not affect the in ltration of CD3-positive T cells and macrophage subsets, including Ly6C hi proin ammatory and CD204 + pro brotic macrophages, or the concentration of proin ammatory cytokines, including IL-2, IL-6, IL-17A , IFN-γ, tumor necrosis factor (TNF) -α, and pro brotic cytokine IL-4, in the in ammatory stage of bleomycin-injected skin. Therefore, the major antibrotic effects of LG283 in bleomycin-injected skin do not appear to be mediated via suppression of in ammatory cell in ltration or subsequent cytokine production.
Since TGF-β/Smad3 signaling has been considered to contribute to the development of bleomycininduced skin brosis (8-11, 37, 38), we evaluated the effect of LG283 on this signaling cascade. Although LG283 did not affect the expression of TGF-β1, increased expression of p-Smad3 was signi cantly inhibited by LG283 treatment in the bleomycin-injected skin. Interestingly, the expression of Snail1 and/or 2 was increased in skin cells including epidermal keratinocytes, endothelial cells, and macrophages following bleomycin injection, an effect inhibited by LG283 treatment. These ndings were similar to that of in vitro investigation of TGF-β-stimulated broblasts and epithelial cells, and cytokine cocktail (TGF-β2, TNF-α, IL-1β)-stimulated endothelial cells. Therefore, our ndings indicate that disrupted TGFβ/Smad/Snail signaling may ameliorate skin brosis and vascular injury via antagonizing the differentiation of resident broblasts, epithelial cells, and endothelial cells into myo broblasts.
A few points remain unclear and future studies will be required to address these issues. The effects of LG283 on skin brosis have only been studied in one animal model. Additionally, the anti-brotic and antivasculopathic effects of LG283 in other organs will need to be investigated in animal models. Regarding in vitro experiments, the effects of LG283 on EMT and EndoMT should also be con rmed in other epithelial and endothelial cell lines. Thus, additional preclinical investigations will be required and the more precise safety pro le of this compound will be needed to determined prior to use of LG283 in SSc clinical trials.

Conclusions
This study demonstrates that the small compound LG283 may be effective for treatment of skin brotic disorders via inhibition of Smad3 phosphorylation and consequent Snail1 and 2 expression downstream of the TGF-β receptor. As a result, the differentiation of various precursors into myo broblasts and subsequent tissue brosis and/or vascular injury are inhibited (Fig. 8). We propose that the screening of EMT and/or EndoMT regulatory compounds may result in additional therapeutic approaches for SSc treatment. The animal protocols of this study were approved by the Committee on Animal Experimentation of University of Fukui (Number 29048).

Consent for publication
Not applicable.
Availability of data and materials LG283 inhibits the brogenic activity of cultured human dermal broblasts stimulated with TGF-β1.
Human broblasts were pretreated with DMSO or various concentrations of DMSO-diluted LG283 for 1 hour, followed by stimulation with 10 ng/ml human rTGF-β1 for additional 24 h. (a and b) After harvest, mRNA and protein expression of COL1A2 and FN-1 were evaluated by real-time RT-PCR and western blot, respectively. Values were normalized to GAPDH levels and are shown as the mean of fold change compared to vehicle control ± SEM. All values represent mean ± SEM; n = 5 each group; *, p ≤ 0.05. (c) Effects of LG283 on mRNA expression of transcription factors associated with mesenchymal transition in human dermal broblasts. All values represent mean ± SEM; n = 5 each group; *, p ≤ 0.05. (d) Human dermal broblasts were immunostained for phospho Smad3 (p-Smad3, green). Representative images are shown on the left (40-fold magni cation). All values represent mean ± SEM; n = 5 each group; **, p ≤ 0.01. Figure 2 LG283 antagonizes EMT in vitro. The effect on EMT in NanoCulture Plate (NCP). Human lung carcinoma epithelial cell line A549 cells were grown in either 2-D or 3-D NCP conditions with or without rTGF-β2 only or TGF-β2-plus LG283. (a) Representative images of 2-D cultured A549 cells. The cells exhibited a round, con uent epithelioid appearance irrespective of the presence (right upper) or absence of LG283 (left upper). Treatment with rTGF-β2 for 72 h resulted in a morphological change to broblastic spindle shape (left lower), which was blocked by co-treatment with LG283 (right lower). (b) Representative images of 3-D nanocultured A549 cells. The cells rapidly colonized and grew similarly in the absence or presence of LG283 (right or left lower, respectively). Treatment with rTGF-β2 for 72 h induced decolonization with poor intercellular junction formation and decreased colony size (left lower). Both of these effects were blocked by co-treatment with LG283 (right lower).  LG283 antagonizes EndoMT in vitro. HUVEC cells were exposed to a combination of cytokines (TGF-β2 at 2.5 ng/ml, TNF-α at 1.0 ng/ml, and IL-1 at 2.0 ng/ml) in experimental medium for the indicated interval or 48 h, in the presence or absence of LG283 (0.5 μM). Expression of an endothelial marker (CD31), mesenchymal marker (FN-1) (a), and transcription factors associated with EndoMT (SNAIL1, 2, and ZEB1, 2) (b) were quanti ed with real-time qRT-PCR. Values were normalized to GAPDH levels and are shown as the mean of the fold change compared to vehicle control ± SD of three independent experiments. All values represent mean ± SEM; n =4 each group; **, p ≤ 0.01.  The effect of oral LG283 on expression of cytokines and Smad3. Crude lysate and mRNA were extracted from skin samples of placebo-and LG283-treated mice at day 7 after bleomycin injection. (a) Concentration of the indicated cytokines was examined by cytometric bead array. Values represent mean ± SEM; n=5 each group; **, p< 0.01. (b and c) TGF-β1 mRNA and protein as evaluated with real-time RT-PCR of the skin and cytometric bead array of the skin extracts, respectively. Values represent mean ± SEM; n = 5 each group. (d) Protein expression levels of Smad3 and p-Smad3 were quanti ed using western blotting. Values represent mean ± SEM; n = 5 each group; **, p< 0.01.

Figure 6
The effect of oral LG283 on the expression of transcription factors associated with EMT and EndoMT in bleomycin-injected mouse skin. (a) mRNA expression of transcription factors in skin samples of placeboand LG283-treated mice at day 7 after bleomycin injection were quantitatively analyzed by real-time RT-PCR. Values represent mean ± SEM; n = 5 each group; **, p< 0.01. (b) Skin sections were immunostained for SNAIL1 and 2. Scale bar, 100 μm. Representative immunohistochemistry images are shown. n = 5 each group.

Figure 8
Putative mechanism of LG283-mediated inhibition of skin brosis.