SB431542 Research Articles

ALK5 Inhibition of Subconjunctival Scarring From Glaucoma Surgery: Effects of SB-431542 Compared to Mitomycin C in Human Tenon's Capsule Fibroblasts.

The gold standard for managing postoperative ocular fibrosis in glaucoma surgery is the chemotherapeutic mitomycin C (MMC) despite its association with significant adverse effects. This study compares in vitro the antifibrotic efficacy and cytotoxicity of the small-molecule TGFβ1 inhibitor SB-431542 (SB) to MMC. To measure collagen contraction, human Tenon's capsule fibroblasts (HTCFs) embedded in a three-dimensional collagen lattice were exposed to 0.2 mg/mL MMC or 20 µM SB followed by incubation with 2 ng/mL TGFβ1. Total protein extracted from experimentally treated HTCFs underwent immunoblotting for α-smooth muscle actin (α-SMA), matrix metallopeptidase 9 (MMP-9), and EDA splice-variant fibronectin (EDA-FN) expression. Cytotoxicity and cell metabolism were assessed using LIVE/DEAD staining, lactate dehydrogenase (LDH) assay, and methylthiazole tetrazolium (MTT) assay. Collagen lattice contraction in TGFβ1-induced HTCFs was significantly lowered by SB and MMC. Pretreatment with SB and MMC significantly lowered protein expression of α-SMA, MMP-9, and EDA-FN in HTCFs relative to TGFβ1 alone. HTCF viability in collagen lattices was significantly reduced with MMC pretreatment but not SB pretreatment. MMC-pretreated HTCFs had a significant increase in LDH release after 3 hours and a decrease in MTT activity after 20 minutes, while SB-pretreated HTCFs showed no significant changes via MTT or LDH assay during the same treatment period. SB shows comparable efficacy to MMC in reducing expression of fibrosis-promoting proteins in HTCFs and in vitro scarring activity. SB distinguishes itself from MMC by exhibiting less cytotoxicity in both two-dimensional and three-dimensional in vitro assays. This study demonstrates in vitro the potential of SB as a safer alternative ocular antifibrotic agent.

3D Bioprinting Mesenchymal Stem Cell-Derived Neural Tissues Using a Fibrin-Based Bioink.

Current treatments for neurodegenerative diseases aim to alleviate the symptoms experienced by patients; however, these treatments do not cure the disease nor prevent further degeneration. Improvements in current disease-modeling and drug-development practices could accelerate effective treatments for neurological diseases. To that end, 3D bioprinting has gained significant attention for engineering tissues in a rapid and reproducible fashion. Additionally, using patient-derived stem cells, which can be reprogrammed to neural-like cells, could generate personalized neural tissues. Here, adipose tissue-derived mesenchymal stem cells (MSCs) were bioprinted using a fibrin-based bioink and the microfluidic RX1 bioprinter. These tissues were cultured for 12 days in the presence of SB431542 (SB), LDN-193189 (LDN), purmorphamine (puro), fibroblast growth factor 8 (FGF8), fibroblast growth factor-basic (bFGF), and brain-derived neurotrophic factor (BDNF) to induce differentiation to dopaminergic neurons (DN). The constructs were analyzed for expression of neural markers, dopamine release, and electrophysiological activity. The cells expressed DN-specific and early neuronal markers (tyrosine hydroxylase (TH) and class III beta-tubulin (TUJ1), respectively) after 12 days of differentiation. Additionally, the tissues exhibited immature electrical signaling after treatment with potassium chloride (KCl). Overall, this work shows the potential of bioprinting engineered neural tissues from patient-derived MSCs, which could serve as an important tool for personalized disease models and drug-screening.

Induction of cyclophilin A by influenza A virus infection facilitates group A Streptococcus coinfection.

During influenza A epidemics, bacterial coinfection is a major cause of increased morbidity and mortality. However, the roles of host factors in regulating influenza A virus (IAV)-triggered bacterial coinfection remain elusive. Cyclophilin A (CypA) is an important regulator of infection and immunity. Here, we show that IAV-induced CypA expression facilitates group A Streptococcus (GAS) coinfection both invitro and invivo. Upon IAV infection, CypA interacts with focal adhesion kinase (FAK) and inhibited E3 ligase cCbl-mediated, K48-linked ubiquitination of FAK, which positively regulates integrin α5 expression and actin rearrangement via the FAK/Akt signaling pathway to facilitate GAS colonization and invasion. Notably, CypA deficiency or inhibition by cyclosporine A significantly inhibits IAV-triggered GAS coinfection in mice. Collectively, thesefindings reveal that CypA is critical for GAS infection, and induction of CypA expression is another way for IAV to promote bacterial coinfection, suggesting that CypA is a promising therapeutic target for the secondary bacterial infection.

Activin/Nodal/TGF-β Pathway Inhibitor Accelerates BMP4-Induced Cochlear Gap Junction Formation During in vitro Differentiation of Embryonic Stem Cells.

Mutations in gap junction beta-2 (GJB2), the gene that encodes connexin 26 (CX26), are the most frequent cause of hereditary deafness worldwide. We recently developed an in vitro model of GJB2-related deafness (induced CX26 gap junction-forming cells; iCX26GJCs) from mouse induced pluripotent stem cells (iPSCs) by using Bone morphogenetic protein 4 (BMP4) signaling-based floating cultures (serum-free culture of embryoid body-like aggregates with quick aggregation cultures; hereafter, SFEBq cultures) and adherent cultures. However, to use these cells as a disease model platform for high-throughput drug screening or regenerative therapy, cell yields must be substantially increased. In addition to BMP4, other factors may also induce CX26 gap junction formation. In the SFEBq cultures, the combination of BMP4 and the Activin/Nodal/TGF-β pathway inhibitor SB431542 (SB) resulted in greater production of isolatable CX26-expressing cell mass (CX26+ vesicles) and higher Gjb2 mRNA levels than BMP4 treatment alone, suggesting that SB may promote BMP4-mediated production of CX26+ vesicles in a dose-dependent manner, thereby increasing the yield of highly purified iCX26GJCs. This is the first study to demonstrate that SB accelerates BMP4-induced iCX26GJC differentiation during stem cell floating culture. By controlling the concentration of SB supplementation in combination with CX26+ vesicle purification, large-scale production of highly purified iCX26GJCs suitable for high-throughput drug screening or regenerative therapy for GJB2-related deafness may be possible.

Photo-activated chemo-immunotherapy for metastatic cancer using a synergistic graphene nanosystem

We developed a novel treatment strategy for metastatic cancer by synergizing photothermal therapy (PTT), chemotherapy, and immunotherapy through a nanosystem to trigger host antitumor immunity. The nanosystem was constructed by loading mitoxantrone (MTX), a chemotherapeutic agent, and SB-431542 (SB), a transforming growth factor beta (TGF-β) inhibitor, onto reduced graphene oxide (rGO). Intratumoral administration of rGO/MTX/SB, followed by non-invasive irradiation of a near-infrared laser, destroyed local primary tumors and inhibited distant metastases in 4T1 mouse mammary tumor model, which is poorly immunogenic and highly metastatic. After treatment, 70% of the tumor-bearing mice became long-term survivors and developed a tumor type-specific immunity to resist rechallenged tumor cells. We found that rGO-based PTT provided an immunogenic antigen source, forming in situ vaccination with rGO as an immune-adjuvant. The use of SB changed the tumor microenvironment and improved the therapeutic effect of MTX-generated chemotherapy and rGO-based PTT. The immunological functions of MTX, SB, and rGO acted synergistically to induce an effective tumor vaccination, as evidenced by the increased infiltration of tumor-specific cytotoxic CD8+ T lymphocytes and decreased infiltration of regulatory T cells (Tregs) in distal tumors. Collectively, we demonstrated that rGO/MTX/SB combined with laser irradiation provided a synergistic chemo-immuno-photothermal effect against tumors by in situ vaccination and inhibition of immunosuppressive microenvironment. This unique combination embodies a promising approach to treat metastatic cancers by inducing a systemic antitumor response through a local intervention.

Induced Fetal Human Muscle Stem Cells with High Therapeutic Potential in a Mouse Muscular Dystrophy Model.

SummaryDuchenne muscular dystrophy (DMD) is a progressive and fatal muscle-wasting disease caused by DYSTROPHIN deficiency. Cell therapy using muscle stem cells (MuSCs) is a potential cure. Here, we report a differentiation method to generate fetal MuSCs from human induced pluripotent stem cells (iPSCs) by monitoring MYF5 expression. Gene expression profiling indicated that MYF5-positive cells in the late stage of differentiation have fetal MuSC characteristics, while MYF5-positive cells in the early stage of differentiation have early myogenic progenitor characteristics. Moreover, late-stage MYF5-positive cells demonstrated good muscle regeneration potential and produced DYSTROPHIN in vivo after transplantation into DMD model mice, resulting in muscle function recovery. The engrafted cells also generated PAX7-positive MuSC-like cells under the basal lamina of DYSTROPHIN-positive fibers. These findings suggest that MYF5-positive fetal MuSCs induced in the late stage of iPSC differentiation have cell therapy potential for DMD.

Temporal Modulations of NODAL, BMP, and WNT Signals Guide the Spatial Patterning in Self-Organized Human Ectoderm Tissues

Summary Developmental biology studies using model organisms suggest that the emergence of spatial patterning in the ectoderm is mediated by the morphogen gradients. However, it is still unclear whether the morphogen gradient is necessary and dominates the cell spatial patterning, particularly in human genetic background. Here, we demonstrate that human pluripotent stem cells can self-organize to concentric rings of all major cell types in ectoderm when cultured on micropatterned surfaces in a chemically defined condition. We reveal that modulating the dynamics of NODAL, BMP, and WNT signals is sufficient to control the spatial order of different cell types. Our mathematical model suggests that changes in wavelength and phase of signaling patterns formed via reaction-diffusion may be the mechanism by which temporal information is translated into spatial information. Together, our work demonstrates that in vitro human ectoderm microtissues have great potential in understanding the mechanisms of early-stage human development.

Olig2 positive cells derived from hair follicle neural crest stem cells in rats

Motor neuron disease (MND) is a kind of common clinical nervous system disease with typical characteristic of progressive motor neurons degeneration or death. Motor neuron derived from stem cells or motor neuron progenitor cells will be a good choice to be used for treatment of the disease. In this study, we used the combination of 5 small molecular including CHIR99021 (CHIR), SB431542 (SB), DMH1 (DMH), retinoic acid (RA) and Purmorphamine (Pur) to induce hair follicles neural crest stem cells (hfNCSCs) to motor neurons progenitors (MNPs). Valproic acid (VPA) was used to make MNPs proliferation. RA and Pur were used to try to induce MNPs toward motor neurons (MNs) and CpdE was tried for MNs maturation. Nestin, β-tubulin Ш (Tuj1), microtubule associated protein 2 (MAP2), Olig2, choline acetyltransferase (ChAT)and TUBB3 were examined at protein and mRNA levels by immunofluoresence cytochemistry, western blot and real time PCR at 6, 16 and 22 days. Our data showed cells changed into bipolar or multipolar shape forming the cell clusters like scattered rosettes. Nestin expression decreased significantly at 22 days. Compared to 6 days, percentage of Olig2 + MNPs was higher, (88.53 ± 6.67)%, and Olig2 expression at protein and gene level was lower at 22 days. Percentage of MAP2 positive cells increased to (90.62 ± 2.31) % and ChAT positive cells increased to (83.29 ± 6.62) % at 22 days. But no expression of ChAT was examined by western blot and real time PCR. It indicates that these 5 molecular can differentiate hfNCSCs into Olig2 positive cells with a unipotent differentiation toward motor neurons.

The Interaction Between Nodal, Hypoxia-Inducible Factor 1 Alpha, and Thrombospondin 1 Promotes Luteolysis in Equine Corpus Luteum.

The regulation of corpus luteus (CL) luteolysis is a complex process involving a myriad of factors. Previously, we have shown the involvement of Nodal in functional luteolysis in mares. Presently, we ask the extent of which Nodal mediation of luteolysis is done through regulation of angioregression. We demonstrated the interaction between Nodal and hypoxia-inducible factor 1 α (HIF1α) and thrombospondin 1/thrombospondin receptor (TSP1/CD36) systems, could mediate angioregression during luteolysis. First, we demonstrated the inhibitory effect of Nodal on the vascular marker platelet/endothelial cell adhesion molecule 1 (CD31). Also, treatment of mid CL explants with vascular endothelial growth factor A (VEGFA) showed a trend on activin-like kinase 7 (Alk7) protein inhibition. Next, Nodal was also shown to activate HIF1α and in vitro culture of mid CL explants under decreased oxygen level promoted Nodal expression and SMAD family member 3 (Smad3) phosphorylation. In another experiment, the crosstalk between Nodal and TSP1/CD36 was investigated. Indeed, Nodal increased the expression of the anti-angiogenic TSP1 and its receptor CD36 in mid CL explants. Finally, the supportive effect of prostaglandin F2α (PGF2α) on TSP1/CD36 was blocked by SB431542 (SB), a pharmacological inhibitor of Nodal signaling. Thus, we evidenced for the first time the in vitro interaction between Nodal and both HIF1α and TSP1 systems, two conserved pathways previously shown to be involved in vascular regression during luteolysis. Considering the given increased expression of Nodal in mid CL and its role on functional luteolysis, the current results suggest the additional involvement of Nodal in angioregression during luteolysis in the mare, particularly in the activation of HIF1α and TSP1/CD36.

Growth differentiation factor 8 regulates SMAD2/3 signaling and improves oocyte quality during porcine oocyte maturation in vitro†.

Growth differentiation factor 8 (GDF8), also known as myostatin, is a member of the transforming growth factor-β (TGF-β) family and has been identified as a strong physiological regulator of muscle differentiation. Recently, the functional role of GDF8 in reproductive organs has received increased interest following its detection in the human placenta and uterus. To investigate the effects of GDF8 during porcine oocyte in vitro maturation (IVM), we assessed the quality of matured oocytes. Furthermore, we investigated the specific gene transcription and protein activation levels in oocytes and cumulus cells after IVM and subsequent embryonic development after in vitro fertilization and parthenogenetic activation. Prior to these experiments, the concentration of GDF8 in porcine follicular fluid was determined. During the entire IVM period, 1.3 ng/mL GDF8 and its signaling inhibitor SB431542 (SB) at 5 μM were added as control, SB, SB+GDF8, and GDF8 groups, respectively. Our results demonstrate that supplementation with GDF8 during porcine oocyte IVM enhanced both meiotic and cytoplasmic maturation, with altered transcriptional patterns, via activation of Sma- and Mad-related protein 2/3 (SMAD2/3). Using the pharmacological inhibitor SB431542, we demonstrated that inhibition of GDF8-induced Smad2/3 signaling reduces matured oocyte quality. In conclusion, for the first time, we demonstrated paracrine factor GDF8 in porcine follicular fluid in vivo. Furthermore, we showed that GDF8 supplementation improved mature oocyte quality by regulating p38 mitogen-activated protein kinase phosphorylation and intracellular glutathione and reactive oxygen species levels during porcine IVM.

Modulation of Mesodermal Patterning Combined with High VEGFA Concentrations Promote Robust Arterial Hemogenic Endothelium Differentiation from Human iPSCs

One of the most promising objectives of clinical hematology is to derive transplantable autologous hematopoietic stem/progenitor cells (HSPCs) from human iPSCs ex vivo, but efficient and clinically relevant methodologies remain unavailable. Observations in the developing embryo indicate that definitive HSPCs arise in the dorsal aorta from hemogenic endothelium (HE) in close association with an arterial vascular endothelial niche. The Notch pathway plays a key role in arterial and HSC differentiation; in the embryo, only HE populations found in arterial regions with active Notch signaling through Delta-like ligand 4 (Dll4) and Jagged 1 (Jag1) lead to HSPCs with repopulating potential. Recent studies have also shown that modulation of mesodermal patterning through repression and activation of Activin/Nodal and Wnt/β-catenin pathways, respectively, promotes arterial programs and definitive hematopoiesis. To facilitate the development of functional HSPCs from human iPSCs, we previously developed a simple, monolayer-based, chemically-defined, and scalable differentiation protocol requiring no replating or embryoid body (EB) formation (commercially available as STEMdiffTM Hematopoietic Kit, Stem Cell Technologies). During the first 3 days, mesodermal specification is induced using morphogens (bFGF, BMP4, VEGF 10ng/mL) and, for the subsequent 18 days, cells are further differentiated into HSPCs with the addition of hematopoietic cytokines (SCF, Flt3L and TPO). As previously presented by our group, this differentiation system recapitulates the successive waves of hematopoiesis during development and leads to robust production of immunophenotypic HSC-like cells (CD34+CD38-CD90+CD45RA-CD49f+). However, these cells do not result in efficient, long-term engraftment in immunodeficient (NSG) mouse models. Characterization of the supportive monolayer from which HSPCs arise during vitro differentiation revealed limited percentages of arterial HE (CD43-CD45-CD34hiCD144+CD73-Dll4+) and arterial endothelium (CD43-CD45-CD34hiCD144+CD73midCD184+), and overabundance of stromal cells (CD43-CD45-CD34-CD144-) which upregulate MSC markers CD105, CD73, and CD90 at later stages in culture. This provides a possible explanation for the lack of engraftment potential of iPSC-derived HSPCs in this system (Panel A). To restrict stromal development and further promote differentiation and maintenance of a supportive arterial endothelial niche, we modified the standard differentiation protocol by addition of CHIR99021 (CHIR) and SB431542 (SB) during the mesodermal stage of differentiation (days 2-3) to activate Wnt/β-catenin and block of Activin/Nodal signaling, respectively. Given that VEGF acts upstream of the Notch pathway during arterial endothelial differentiation, we also increased the concentration of VEGFA 20-fold throughout differentiation (200ng/mL). Our results showed that mesodermal patterning alone (CHIR/SB) activated critical HoxA cluster genes in both early endothelial and late HSPC populations but was insufficient to repress stromal production and maintain an endothelial niche beyond early culture days. However, increased VEGF concentrations, alone or in combination with CHIR/SB, markedly reduced stromal differentiation (Panel B) and enhanced arterial endothelium formation (Panel C) compared to the standard system (control). Importantly, combination treatments also led to significantly higher percentages of arterial HE at days 5 and 7 (panel D). Current assessment of these treatments on the hematopoietic potential of the system is ongoing, and include NSG mouse transplantations. Overall, our data indicate that commercially available technologies can be further modified and improved to move closer to chemically-defined and scalable HSPC differentiation protocols. DisclosuresLarochelle:Stem Cell Technologies: Patents & Royalties: StemDiff Hematopoietic Differentiation Kit.

TGFB1 modulates in vitro secretory activity and viability of equine luteal cells

In the present report we describe the involvement of transforming growth factor B1 (TGF) in functional regression and structural luteolysis in the mare. Firstly, TGF and its receptors activin-like kinase (ALK) 5 and TGF receptor 2 were identified in corpus luteum (CL) steroidogenic, endothelial and fibroblast-like cells. Also, TGF and ALK5 protein expression were shown to be increased in Mid-, and Late-CL (p < 0.05). Subsequently, using an in vitro model with Mid-CL cells, we studied the role of TGF on secretory activity and cell viability. Cell treatment with TGF decreased progesterone (P4) and prostaglandin (PG) E2 concentrations in culture media (p < 0.05), and downregulated mRNA and protein of StAR, CYP11A1, cPGES and mPGES1 (p < 0.05). Conversely, TGF augmented PGF2a concentration in culture media, through PTGS2 and PGFS gene expression activation (p < 0.05). When cells were incubated with PGF2a, both TGF and ALK5 were upregulated (p < 0.05). Additionally, treatment with the pharmacological inhibitor of ALK5, ALK4 and ALK7 - SB431542 (SB) attenuated PGF2a functional and structural luteolytic actions. Indeed, SB blocked: (i) PGF2a inhibitory effect on StAR, CYP11A1, 3BHSD and mPGES1; (ii) PGF2a auto-amplification signal via PTGS2 and PGFS expression (p < 0.05); (iii) the PGF2a-induced BAX and FASL expression (p < 0.05). Finally, TGF decreased cell viability (p < 0.05) and promoted caspase 3 activity (p = 0.08) and the expression of pro-apoptotic FASL and BAX (p < 0.05). Our results suggest that TGF supports functional regression and structural luteolysis, and also confirm the importance of ALK5, ALK4 and ALK7 activation during PGF2a mediated luteolysis in mares.

GP73 promotes epithelial-mesenchymal transition and invasion partly by activating TGF-β1/Smad2 signaling in hepatocellular carcinoma.

The transforming growth factor-β1 (TGF-β1) signaling pathways contribute to cell metastasis and epithelial-mesenchymal transition (EMT). Golgi protein 73 (GP73), a type II transmembrane protein in the Golgi apparatus, was initially regarded as a potential biomarker for the diagnosis of hepatocellular carcinoma (HCC). Recently, it was reported that GP73 acts as a key oncogene by promoting HCC growth and metastasis. However, the role of GP73 in metastasis, especially when involving signaling pathways, is uncertain. Here, we report that GP73, which is upregulated in HCC tissues and cell lines, is associated with tumor size, tumor node metastasis stage, distant metastasis and vascular invasion. The ectopic overexpression of GP73 increased HCC cell invasion, EMT and metastasis both in vitro and in vivo. Conversely, GP73 knockdown inhibited invasion and EMT. Moreover, GP73 enhanced p-Smad2 and p-Smad3 levels by mediating TGF-β1, thus leading to the promotion of EMT and invasion in HCC cells. In contrast, we used SB431542 (SB) to repress p-Smad2 and p-Smad3 expression, which resulted in a reversion of EMT. Furthermore, when the TGF-β1/Smad pathway was blocked, upregulation of GP73 still caused an enhanced EMT and invasion, and downregulation of GP73 resulted in a decreased in EMT and invasion. In clinical HCC samples, GP73 positively correlated with TGF-β1/Smad2, which was upregulated in HCC. Taken together, our findings highlight the important role of GP73 in regulating EMT and metastasis in HCC partly by targeting TGF-β1/Smad2 signaling, suggesting that GP73 may represent a novel potential therapeutic target and prognostic marker for the treatment and diagnosis of HCC.

Blocking/Knocking out Smad3 Alleviates Doxorubicin Effects on Endothelial Cells

Recent studies have shown that patients with early-stage cancers are now more likely to die from complications of anticancer therapy than cancer. Doxorubicin (Dox), a drug used to treat numerous cancers, is known to cause delayed cardiomyopathy. We have previously reported reduced microvascular density and increased levels of TGFbeta related transcripts in Dox treated mouse hearts. We hypothesized that microvascular remodeling in Dox treated hearts is mediated by enhanced TGFbeta/Smad3 signaling. We observed the inhibition of human umbilical vein endothelial cell (HUVEC) proliferation and suppression of the formation of vessel-like structures by Dox. TGFbeta pathway inhibitor SB431542 (SB), which selectively blocks Smad2/3 signaling, alleviated the effects of Dox in vitro. In aortic ring experiments, Dox decreased the number of sprouts in aortas from wild type mice while aortic preparations from Smad3 knockout animals were more resistant to the deleterious effect of Dox and produced increased numbers of sprouts. In experiments with co-cultures of cardiac fibroblasts and HUVEC, we identified the detachment of endothelial cells as an additional putative mechanism of microvascular remodeling by Dox. Live imaging of co-cultures treated with Dox in the presence of caspase-3/7 substrate revealed that detaching endothelial cells were not undergoing apoptosis prior to detachment. SB prevented detachment and increased the number of endothelial cells in co-cultures treated with Dox. Treatment with Dox led to the reduced adhesion of endothelial cells to laminin, a major constituent of the endothelial basement membrane while SB improved adhesion of Dox treated cells to laminin. In conclusion, Dox causes inhibition of sprouting angiogenesis in aortic rings and detachment of endothelial cells in an in vitro co-culture model of formation of vessel-like structures. These deleterious processes appear to be mediated by TGFbeta/Smad3 signaling as they were alleviated by both a pathway inhibitor and a genetic Smad3 knockout.

Roles of TGF\u03b2 and FGF signals during growth and differentiation of mouse lens epithelial cell in vitro

Transforming growth factor β (TGFβ) and fibroblast growth factor (FGF) signaling pathways play important roles in the proliferation and differentiation of lens epithelial cells (LECs) during development. Low dosage bFGF promotes cell proliferation while high dosage induces differentiation. TGFβ signaling regulates LEC proliferation and differentiation as well, but also promotes epithelial-mesenchymal transitions that lead to cataracts. Thus far, it has been difficult to recapitulate the features of germinative LECs in vitro. Here, we have established a LEC culture protocol that uses SB431542 (SB) compound to inhibit TGFβ/Smad activation, and found that SB treatment promoted mouse LEC proliferation, maintained LECs’ morphology and distinct markers including N-cadherin, c-Maf, Prox1, and αA-, αB-, and β-crystallins. In contrast, low-dosage bFGF was unable to sustain those markers and, combined with SB, altered LECs’ morphology and β-crystallin expression. We further found that Matrigel substrate coatings greatly increased cell proliferation and uniquely affected β-crystallin expression. Cultured LECs retained the ability to differentiate into γ-crystallin-positive lentoids by high-dosage bFGF treatment. Thus, a suppression of TGFβ/Smad signaling in vitro is critical to maintaining characteristic features of mouse LECs, especially expression of the key transcription factors c-Maf and Prox1.

Methotrexate-Induced Epithelial-Mesenchymal Transition in the Alveolar Epithelial Cell Line A549.

Methotrexate (MTX) therapy of certain cancers and rheumatoid arthritis often induces serious interstitial lung complications including pulmonary fibrosis. In this study, we investigated the epithelial-mesenchymal transition (EMT) induced by MTX and by transforming growth factor (TGF)-β1 in the human alveolar epithelial cell line A549 in order to develop new strategies for the prevention of EMT. First, we examined the effect of TGF-β1 and MTX on cell morphology and the expression of EMT-related mRNAs in A549 cells. Then, the effects of SB431542 (SB), a potent inhibitor of TGF-β receptor kinase, and a neutralizing antibody for TGF-β1 on the phenotypic changes of A549 cells induced by TGF-β1 and MTX were examined. After incubation with TGF-β1 and MTX, the mRNA expression of epithelial markers such as cytokeratin 19 was reduced, while that of mesenchymal markers such as α-smooth muscle actin was increased. SB suppressed the development of morphological changes and partially rescued alterations in mRNA expression of EMT markers induced by MTX. In addition, the enhancement of SMAD2 phosphorylation by MTX was also prevented by SB. On the other hand, EMT-related changes induced by MTX were not affected by a neutralizing antibody for TGF-β1. We have demonstrated that phenotypic changes of A549 cells induced by MTX are partly mediated by a TGF-β1-related intracellular signaling pathway, although TGF-β1 itself is not directly involved in this process.

Effect of TGF-β1 on the Migration and Recruitment of Mesenchymal Stem Cells after Vascular Balloon Injury: Involvement of Matrix Metalloproteinase-14.

Restenosis or occlusion after vascular procedures is ascribed to intimal hyperplasia. Transforming growth factor (TGF)-β1 is involved in recruitment of mesenchymal stem cells (MSCs) following arterial injury, and its release from latent TGF-binding protein by matrix metalloproteinase (MMP)-14-induced proteolysis contributes to neointima formation. However, the relationship between MMP-14 and TGF-β1 activation in restenosis is unknown. This study investigated the relationship using a rat model of balloon-induced injury. Rats were assigned to vehicle-, SB431542 (SB)-, or recombinant human (rh)TGF-β1-treated groups and examined at various time points after balloon-induced injury for expression of TGF-β1/Smad signalling pathway components, MMP-14 and MSCs markers including Nestin, CD29, and Sca1+CD29+CD11b/c−CD45−. Intimal hyperplasia was reduced in SB- and rhTGF-β1-treated rats. The expression of TGF-β1, TGF-β1RI, and Smad2/3 was decreased, but the levels of phosphorylated Smad2/3 were higher in SB-treated rats than vehicle-treated after 7 days to 14 days. rhTGF-β1 administration decreased the expression of TGF-β1/Smad pathway proteins, except for TGF-β1RI. Nestin and CD29 expression and the number of Sca1+CD29+CD11b−CD45− cells were reduced, whereas MMP-14 expression was increased after SB431542 and rhTGF-β1 administration. These results suggest that TGF-β1/Smad signalling and MMP-14 act to recruit MSCs which differentiate to vascular smooth muscle cells and mesenchymal-like cells that participate in arterial repair/remodelling.

Transient Suppression of TGFβ Receptor Signaling Facilitates Human Islet Transplantation.

Although islet transplantation is an effective treatment for severe diabetes, its broad application is greatly limited due to a shortage of donor islets. Suppression of TGFβ receptor signaling in β-cells has been shown to increase β-cell proliferation in mice, but has not been rigorously examined in humans. Here, treatment of human islets with a TGFβ receptor I inhibitor, SB-431542 (SB), significantly improved C-peptide secretion by β-cells, and significantly increased β-cell number by increasing β-cell proliferation. In addition, SB increased cell-cycle activators and decreased cell-cycle suppressors in human β-cells. Transplantation of SB-treated human islets into diabetic immune-deficient mice resulted in significant improvement in blood glucose control, significantly higher serum and graft insulin content, and significantly greater increases in β-cell proliferation in the graft, compared with controls. Thus, our data suggest that transient suppression of TGFβ receptor signaling may improve the outcome of human islet transplantation, seemingly through increasing β-cell number and function.

The TGF-β pathway mediates doxorubicin effects on cardiac endothelial cells

Elevated ALK4/5 ligands including TGF-β and activins have been linked to cardiovascular remodeling and heart failure. Doxorubicin (Dox) is commonly used as a model of cardiomyopathy, a condition that often precedes cardiovascular remodeling and heart failure. In 7–8-week-old C57Bl/6 male mice treated with Dox we found decreased capillary density, increased levels of ALK4/5 ligand and Smad2/3 transcripts, and increased expression of Smad2/3 transcriptional targets. Human cardiac microvascular endothelial cells (HCMVEC) treated with Dox also showed increased levels of ALK4/5 ligands, Smad2/3 transcriptional targets, a decrease in proliferation and suppression of vascular network formation in a HCMVEC and human cardiac fibroblasts co-culture assay. Our hypothesis is that the deleterious effects of Dox on endothelial cells are mediated in part by the activation of the TGF-β pathway. We used the inhibitor of ALK4/5 kinases SB431542 (SB) in concert with Dox to ascertain the role of TGF-β pathway activation in doxorubicin induced endothelial cell defects. SB prevented the suppression of HCMVEC proliferation in the presence of TGF-β2 and activin A, and alleviated the inhibition of HCMVEC proliferation by Dox. SB also prevented the suppression of vascular network formation in co-cultures of HCMVEC and human cardiac fibroblasts treated with Dox. Our results show that the inhibition of the TGF-β pathway alleviates the detrimental effects of Dox on endothelial cells in vitro.

208 EFFECT OF GDF8 AND SB-431542 ON PORCINE OOCYTE DURING IN VITRO MATURATION AND SUBSEQUENT EMBRYONIC DEVELOPMENT

Growth differentiation factor-8 (GDF8) is a member of the transforming growth factor-β that has been identified as a strong physiological regulator. SB-431542 (SB) is a specific inhibitor of transforming growth factor-β superfamily type I activin receptor-like kinase (ALK) receptors such as ALK4, ALK5, and ALK7. The purpose of this study is investigation of the effects of GDF8 and SB on porcine oocytes during in vitro maturation and subsequent embryonic development. We first performed ELISA to detect GDF8 concentrations in follicular fluid for each size of follicle; sizes were as follows: small (&lt;3 mm), medium (&gt;3 mm and &lt;6 mm), and large (&gt;6 mm) follicle. After detection of the GDF8 concentration in follicular fluid, we investigated the effect of GDF8 and SB treatment during in vitro maturation (IVM) on nuclear maturation, intracellular glutathione (GSH), and reactive oxygen species (ROS) levels, and embryonic development after IVF and parthenogenetic activation (PA). Data were analysed by ANOVA followed by Duncan using SPSS (Statistical Package for Social Science, IBM, New York, NY, USA) mean ± SEM. The ELISA result showed different concentrations of GDF8 for each grade of follicular fluid: small, 0.479 ng mL–1; medium, 0.668 ng mL–1; and large, 1.318 ng mL–1. During the IVM process, 1.318 ng mL–1 of GDF8 and 5 ng mL–1 of SB were added to the maturation medium as control, SB, SB+GDF8, and GDF8 treatment groups. After 44 h of IVM, GDF8 group (90.4%) showed a significantly higher nuclear maturation rate than control and SB+GDF8 groups (85.4 and 81.7%). The SB group (78.9%) showed significantly reduced nuclear maturation rate compared with control (P &lt; 0.05). The GDF8 treatment group showed a significant decreased intracellular ROS and increased GSH levels compared with other groups (P &lt; 0.05). The SB+GBF8 treatment group showed a significantly better cytoplasmic maturation than the SB treatment group. In the PA embryonic development analysis, the GDF8 treatment group showed a significantly higher blastocyst formation rate compared with other groups (47.9, 37.2, 46.4, and 58.7% respectively; P &lt; 0.05). In the IVF embryonic development analysis, the GDF8 treatment groups showed significantly higher blastocyst formation rate compared with the SB group (28.2 and 42.2%, respectively; P &lt; 0.05). In conclusion, treatment with GDF8 during porcine oocyte IVM improved the embryonic developmental competence via increased cytoplasmic maturation and led to better oocyte maturation from the ALK receptor inhibition by SB.