TGF-β Binding Research Articles

HcFAR, a functional inhibitor of goat TGF-β1 identified from excretory and secretory products of Haemonchus contortus

Haemonchus contortus has developed complexed and multifaceted mechanisms of immune evasion to enable the survival in the host. Generating excretion and secretion products (ESPs) to subvert or suppress the functions of host cytokines is a newly immune regulatory pattern found during recent years. Transforming growth factor-β (TGF-β) has critical immune regulatory functions in nematode infections. In this study, co-immunoprecipitation (co-IP) assay was used to identify the goat TGF-β1 binding proteins from HcESPs. The interaction between TGF-β1 and nematode fatty acid retinoid binding domain containing protein of H. contortus (HcFAR) was analyzed by glutathione S-transferase (GST)-pull down assay. The suppressive effect of rHcFAR on TGF-β1-induced immunoglobulin A (IgA) secretion was observed by co-incubation of rHcFAR and TGF-β1 with goat peripheral blood mononuclear cells (PBMCs). The IgA concentrations were determined using enzyme linked immunosorbent assay (ELISA) kit. Meanwhile, the suppressive effect of rHcFAR on TGF-β1-induced T helper (Th) 9 differentiation was investigated by co-incubation of rHcFAR, TGF-β1 and interleukin (IL)-4 with goats PBMCs. In parallel, IL-4 was replaced by IL-6 to determine the effects on the Th17 differentiation. The transcriptions of IL-9 and IL-17 in PBMCs were then evaluated by real-time PCR. Finally, we found that HcFAR from HcESPs could bind to goat TGF-β1 in vitro. The ELISA results of IgA showed that 40 μg/mL rHcFAR could suppress the IgA secretion of PBMCs induced by TGF-β1. Additionally, rHcFAR (at 10 μg/mL and 20 μg/mL) could inhibit the mRNA transcription of IL-9 induced by TGF-β1 and IL-4. Meanwhile, rHcFAR could also downregulate the transcription of IL-17 induced by TGF-β1 and IL-6 in a dose-dependent manner. These results indicated that HcFAR was a functional inhibitor of goat TGF-β1 and this information may help contribute to understanding of the relationship between the ESPs and host cytokines.

Minimal Consequences of CMAH and DBA/2 Backgrounds on a FKRP Deficient Model.

Muscular dystrophies (MD) are a large group of genetic diseases characterized by a progressive loss of muscle. The Latent TGFβ Binding Protein 4 (LTBP4) in the DBA/2 background and the Cytidine Monophosphate-sialic Acid Hydroxylase (CMAH) proteins were previously identified as genetic modifiers in severe MD. We investigated whether these modifiers could also influence a mild phenotype such as the one observed in a mouse model of Limb-Girdle MD2I (LGMD2I). The FKRPL276I mouse model was backcrossed onto the DBA/2 background, and in separate experiments the Cmah gene was inactivated in FKRPL276I mice by crossing with a Cmah-/- mouse and selecting the double-mutants. The mdx mouse was used as control for these two genome modifications. Consequences at the histological level as well as quantification of expression level by RT-qPCR of genes relevant for muscular dystrophy were then performed. We observed minimal to no effect of the DBA/2 background on the mild FKRPL276I mouse phenotype, while this same background was previously shown to increase inflammation and fibrosis in the mdx mouse. Similarly, the Cmah-/- deletion had no observable effect on the FKRPL276I mouse phenotype whereas it was seen to increase features of regeneration in mdx mice. These modifiers were not observed to impact the severity of the presentation of the mild FKRPL276I model. An interesting association of the CMAH modifier with the regeneration process in the mdx model was seen and sheds new light on the influence of this protein on the dystrophic phenotype.

Endothelial Cells Express Activin A which may serve as an antagonist to TGFβ binding to Activin‐Like Kinase 1 Receptor

Transforming growth factor (TGFβ) family and their receptors have been shown to be involved in angiogenesis as well as tumor cells. Members of the family that have been reported to act on endothelial cells are TGFβ, Bone Morphogenetic protein 9 and 10 (BMP‐9, BMP‐10), and Activin A. They all can signal through the type I receptor Activin‐Like Kinase 1 (ALK‐1). We have screened endothelial cells vs tumor cells for ALK‐1, Endoglin, and potential ligands BMP‐10 and Activin A to determine which ligands and receptors may be active in signaling in angiogenesis. We show that endothelial cells express ALK‐1 and endoglin as well as BMP‐10 and Activin A by quantitative RT‐PCR. Transfecting Human Umbilical Vein Endothelial Cells (HUVEC) compared to human 293 cells and Hela cells with an ALK‐1 plasmid resulted in increased expression of ALK‐1 in all cell lines. ALK‐1 overexpression did not affect Smad 1 or Smad 5 expression but increased BMP‐10 and Activin A expression in HUVEC. This data suggests these factors may function as endogenous ligands to control endothelial function and angiogenesis. TGFβ can only bind ALK‐1 on endothelial cells at 37°C but not 23 or 4° C. But our results indicate that endogenous Activin A is bound to ALK‐1 and can be displaced by added TGFβ. This supports the finding above that HUVEC express Activin A. Thus Activin A may function as an endogenous regulator of TGFβ function in endothelial cells.Support or Funding InformationResearch support was from an SFSC Innovation Grant (MP), Portz National Honors Fellowships (GW and NM), and funds from Saint Leo University (ID).

Mathematical modelling of the role of Endo180 network in the development of metastatic bone disease in prostate cancer

Metastatic bone disease (MBD) is a common complication of advanced cancer and recent research suggests that Endo180 expression is dysregulated through the TGFβ-TGFβR-SMAD2/3 signalling pathway during the invasion of tumour cells in the development of MBD. We here provide a model for the dysregulation of the Endo180 network to demonstrate its vital contribution to bone destruction as well as tumour cell growth. The model consisted of a set of ordinary differential equations and reconstructed variations in the bone cells, resultant bone volume, and biochemical factors involved in the TGFβ-TGFβR-SMAD2/3 signalling pathway over time. The model also investigated the underlying mechanism in which the change of TGFβ affects the TGFβ-TGFβR-SMAD2/3 signalling pathway and the resultant Endo180 expression in osteoblastic and tumour cells. The model links the appearance of tumour cells with the inhibition of TGFβ binding to its receptors on osteoblastic cells, to affect TGFβ-TGFβR-SMAD2/3 signalling and Endo180 expression. Temporal variation in bone cells, bone volume, and the biochemical factors involved in the TGFβ-TGFβR-SMAD2/3 pathway as demonstrated in the model simulations agree with published experimental data. The model can be refined based on further discoveries but allows the influence of Endo180 network dysregulation on bone remodelling in MBD to be established. This model could aid in the development of Endo180 targeted therapies for MBD in the future.

Phlorofucofuroeckol A from Ecklonia cava ameliorates TGF-β1-induced fibrotic response of human tracheal fibroblasts via the downregulation of MAPKs and SMAD 2/3 pathways inactivated TGF-β receptor

The objective of this study was to investigate inhibitory effects of a bioactive compound isolated from Ecklonia cava on fibrotic responses to transforming growth factor-β1 (TGF-β1)-stimulated Hs680. Tr human tracheal fibroblasts and the associated mechanisms of action. Post consecutive purification, a potent bioactive compound was identified phlorofucofuroeckol A. Phlorofucofuroeckol A significantly suppressed protein expression levels of collagen type I and α-smooth muscle actin (α-SMA) on TGF-β1-stimulated Hs680. Tr human tracheal fibroblasts. Further mechanistic studies determined that phlorofucofuroeckol A suppressed the phosphorylation of p38, extracellular regulated kinase (ERK), and c-Jun N-terminal kinase (JNK) and SMAD 2/3 in TGF-β1-stimulated Hs680. Tr human tracheal fibroblasts. Moreover, we could show that phlorofucofuroeckol A inhibits binding of TGF-β1 to its TGF-β receptor by molecular docking. Based on the results, we propose that phlorofucofuroeckol A suppresses the MAPKs and SMAD 2/3 pathways and relieves cellular fibrotic activities, thus preventing tracheal fibrosis.

TGF-β induced chemoresistance in liver cancer is modulated by xenobiotic nuclear receptor PXR

ABSTRACT Hepatocellular carcinoma appears as an extremely angiogenic solid tumor marked by apoptosis evasion, dysregulated cell cycle and low sensitivity to chemotherapy. TGF-β, a multifunctional cytokine, plays a pleiotropic role in the tumor microenvironment and has implications in cancer drug resistance. The current study provides novel evidence that TGF-β signaling contributes to drug resistance in liver cancer cells by inducing the expression of xenobiotic nuclear receptor PXR. We observed that PXR increases the expression of drug efflux transporters; therefore, accounting for exacerbated drug resistance. Additionally, anti-apoptotic nature of PXR contributes to TGF-β mediated chemoresistance as seen by procaspase-3 and Mcl-1 cellular levels. TGF-β binding to the TGF-β receptor triggers a complex downstream signaling cascade through a non-canonical SMAD-independent ERK pathway that leads to increased PXR expression. Activated ERK activates ETS1 transcription factor which is a critical regulator of endogenous PXR expression in hepatic cells. Loss of function of ETS1 abrogates the TGF-β induced PXR expression. Together these findings indicate that PXR modulates TGF-β induced resistance to chemotherapy in liver cancer cells. This underscores the need for combinatorial approaches with focus on PXR antagonism to improve drug effectiveness in hepatocellular carcinoma. Abbreviations HCC: Hepatocellular Carcinoma; FDA: Food and Drug Administration; TGF-β: Transforming growth factor-β; PXR: Pregnane X receptor; CAR: Constitutive androstane receptor; P-gp/ABCB1: P-glycoproteins/ATP-binding cassette transporter subfamily B member 1; MRP1/ABCC1 and MRP2/ABCC2: Multidrug-resistance associated proteins; BCRP/ABCG2: Breast cancer resistant protein; DMEs: Drug-metabolizing enzymes; CFDA: 5,6-carboxyfluorescein diacetate; ETS1: Transcription factor E26 transformation specific sequence 1.

Epigenetic regulation of lung cancer cell proliferation and migration by the chromatin remodeling protein BRG1

Malignant lung cancer cells are characterized by uncontrolled proliferation and migration. Aberrant lung cancer cell proliferation and migration are programmed by altered cancer transcriptome. The underlying epigenetic mechanism is unclear. Here we report that expression levels of BRG1, a chromatin remodeling protein, were significantly up-regulated in human lung cancer biopsy specimens of higher malignancy grades compared to those of lower grades. Small interfering RNA mediated depletion or pharmaceutical inhibition of BRG1 suppressed proliferation and migration of lung cancer cells. BRG1 depletion or inhibition was paralleled by down-regulation of cyclin B1 (CCNB1) and latent TGF-β binding protein 2 (LTBP2) in lung cancer cells. Further analysis revealed that BRG1 directly bound to the CCNB1 promoter to activate transcription in response to hypoxia stimulation by interacting with E2F1. On the other hand, BRG1 interacted with Sp1 to activate LTBP2 transcription. Mechanistically, BRG1 regulated CCNB1 and LTBP2 transcription by altering histone modifications on target promoters. Specifically, BRG1 recruited KDM3A, a histone H3K9 demethylase, to remove dimethyl H3K9 from target gene promoters thereby activating transcription. KDM3A knockdown achieved equivalent effects as BRG1 silencing by diminishing lung cancer proliferation and migration. Of interest, BRG1 directly activated KDM3A transcription by forming a complex with HIF-1α. In conclusion, our data unveil a novel epigenetic mechanism whereby malignant lung cancer cells acquired heightened ability to proliferate and migrate. Targeting BRG1 may yield effective interventional strategies against malignant lung cancers.

LRRC33 is a novel binding and potential regulating protein of TGF-β1 function in human acute myeloid leukemia cells

Transforming growth factor‑β1 (TGF-β1) is a versatile cytokine. It has context-dependent pro- and anti-cell proliferation functions. Activation of latent TGF-β1 requires release of the growth factor from pro-complexes and is regulated through TGF-β binding proteins. Two types of TGF-β binding partners, latent TGF-β-binding proteins (LTBPs) and leucine-rich-repeat-containing protein 32 (LRRC32), have been identified and their expression are cell specific. TGF-β1 also plays important roles in acute myeloid leukemia (AML) cells. However, the expression of LTBPs and LRRC32 are lacking in myeloid lineage cells and the binding protein of TGF-β1 in these cells are unknown. Here we show that a novel leucine-rich-repeat-containing protein family member, LRRC33, with high mRNA level in AML cells, to be the binding and regulating protein of TGF-β1 in AML cells. Using two representative cell lines MV4-11 and AML193, we demonstrate that the protein expression of LRRC33 and TGF-β1 are correlated. LRRC33 co-localizes and forms complex with latent TGF-β1 protein on the cell surface and intracellularly in these cells. Similar as in other cell types, the activation of TGF-β1 in MV4-11 and AML193 cells are also integrin dependent. We anticipate our study to be a starting point of more comprehensive research on LRRC33 as novel TGF-β regulating protein and potential non-genomic based drug target for AML and other myeloid malignancy.

32P - Delineating the mechanisms of alpha 1-3 fucosyltransferase FUT11 in ovarian cancer

BackgroundOvarian cancer is the most lethal gynecological cancer and the biological mechanisms remains unclear. The prevalence of abnormal fucosylation has been reported to be closely related to cancer progression and prognosis. It was reported that only FUT3 and/or FUT6 in the α 1-3/4 fucosyltransferase subfamily were required for TGF-β-mediated epithelial-to-mesenchymal transition (EMT) in colorectal cancer. However, it was also reported that the α 1-6 fucosyltransferase FUT8 facilitated TGF-β binding to receptor and stimulated the EMT in breast cancer. As an α 1-3 fucosyltransferase distinct from the α 1-3/4 and 1-6 fucosyltransferase subfamily, the role of FUT11 in ovarian cancer development is less understood. MethodsFUT11 was identified as a novel oncogene related with the EMT process in ovarian cancer by bioinformatic analysis and real-time PCR. The transwell chamber assays were used to examine the changes of the invasion and migration ability after FUT11 knocked-down by siRNA treatment in ovarian cancer cells SK-OV-3 and HEY. Western-blot was usd to determine the changes of EMT genes by FUT11 depletion in response to TGF-β 1. Furthermore, expression and location of R-Smads and Co-Smad were analyzed by western blot and immunofluorescence assay, to evaluate the impact of FUT11 depletion on the TGF-β/Smad pathway. ResultsFUT11 was highly expressed in ovarian cancer, and increased FUT11 expression was significantly correlated with poor survival of the ovarian patients. FUT11 depletion impaired the invasion and migration of the ovarian cancer cell lines, through regulation of the TGF-β 1-induced EMT process. These inhibitory effect was due to downregulation of the expression of SMAD2 and SMAD3, as well as prevention of their translocation from cytoplasm to nucleus. Although expression of SMAD4 were slightly affected, their translocation from cytoplasm to nucleus were inhibited by FUT11 depletion. ConclusionsFUT11 promotes invasion and migration of ovarian cancer through regulation of the TGF-β 1-induced EMT process. FUT11 affects the response to TGF-β 1 by regulation of the expression level of R-Smads and Co-Smad, as well as their translocation from cytoplasm to nucleus. Legal entity responsible for the studyBeijing Obstetrics and Gynecology Hospital, Capital Medical University. FundingThe National Natural Science Foundation of China (81502353 & 81672838), Beijing Municipal Science & Technology Commission (No. Z181100001718193), and Beijing Obstetrics and Gynecology Hospital, Capital Medical University (FCYY201713). DisclosureAll authors have declared no conflicts of interest.

MK5-MEDIATED FIBROBLAST FUNCTION AND EXTRACELLULAR MATRIX HOMEOSTASIS: THE DIFFERENCE BETWEEN QUIESCENT FIBROBLASTS AND ACTIVATED MYOFIBROBLASTS

Cardiac fibroblast hyperplasia associated with enhanced matrix production and deposition is a major determinant of post-injury cardiac remodelling. MAP kinase-activated protein kinase-5 (MK5) is expressed in the heart but its physiological role in the heart is just beginning to be understood. Pressure overload-induced collagen expression is reduced in male MK5 haploinsufficient (MK5+/-) mice. Further, following myocardial infarction, both infarct size and scar size are reduced in male MK5+/- mice. Thus, MK5 may play a role in extracellular matrix (ECM) remodeling. This study sought to determine the role of MK5 in cardiac fibroblast function. Ventricular fibroblasts were isolated from male MK5+/+ or MK5-/- mice. When cultured on a rigid substrate, cardiac fibroblasts adopted an activated myofibroblast phenotype associated with increased αSMA expression. Further, MK5-deficient (MK5-/-; siRNA-mediated MK5 knockdown) myofibroblasts showed increased basal levels of collagen 1-α1 (COL1A1) mRNA and type 1 collagen secretion; however, addition of Ang-II failed to evoke a further increase in either. Confocal immunocytofluorescence revealed type 1 collagen immunoreactivity to be diffuse in MK5+/+ myofibroblasts but condensed in the perinuclear region of MK5-deficient myofibroblasts. When maintained on a compliant (8-kPa) substrate, fibroblasts remain quiescent with reduced αSMA expression. The Ang-II-induced increase in COL1A1 mRNA and type 1 collagen secretion was significantly decreased in MK5-deficient fibroblasts when compared with MK5+/+ fibroblasts. Further, MK5-deficiency impaired the ability of fibroblasts to contract collagen gels. Interestingly, pathway-targeted microarrays revealed that MK5-deficiency in myofibroblasts decreased the abundance of transcripts for ECM proteins such as integrin β3, latent TGF-β binding protein 1, thrombospondin 1, hepatocyte growth factor, and interleukin 13. In quiescent fibroblasts, reduced MK5 expression had little effect on the abundance of transcripts for ECM proteins. MK5 is involved in multiple aspects of fibroblast function and ECM dynamics and the role of MK5 is dependent on the activation status of these cells.

SAT-192 LOSS OF MACROPHAGE-SPECIFIC TLR4 INHIBITS OBSTRUCTIVE RENAL FIBROSIS BY BLOCKING MACROPHAGE-TO-MYOFIBROBLAST TRANSITION VIA A BAMBI-DEPENDENT MECHANISM

Macrophages are a key inflammatory cell that plays a critical role in acute and chronic kidney diseases. Toll-like receptor (TLR) 4 is highly expressed on inflammatory macrophages and has been shown to trigger acute renal inflammation. However, the role of macrophage-specific TLR4 in progression of chronic kidney disease remains unclear, which was investigated in the present study in a mouse model of unilateral ureter ligation (UUO). A mouse model of UUO was induced in Tlr4flox/floxand lysozyme Mpromoter-driven cre(LysM-cre) transgenic mice (C57BL/6). Role and mechanisms of TLR4 in macrophage-mediated renal fibrosis were studied. UUO led to remarkably F4/80+macrophage and CD3+/CD4+T cell infiltration and progressive renal fibrosis in Tlr4flox/floxmouse kidney. In contrast, deletion of macrophage-specific TLR4 blunted these inflammatory responses in LysM-cre/Tlr4flox/flox animals. Mechanistically, these inhibitory effects on renal inflammation and fibrosis in LysM-cre/Tlr4flox/floxmice were associated with the shift of macrophages from M1 (F4/80+/iNOS+) to M2 phenotype (F4/80+/CD206+). Most importantly, we also found that deletion of TLR4 from macrophage largely suppressed progressive renal fibrosis by inhibiting macrophage-to-myofibroblast transition (MMT) as identified by F4/80+αSMA+cells. This novel observation suggests that macrophage-TLR4 may trigger inflammatory M1 macrophage activation and subsequent transition to the fibrogenic phenotype via the MMT process. Further studies revealed that BMP and activin membrane-bound inhibitor (Bambi), a TGF-β1 pseudoreceptor that competitively inhibits the binding of active TGF-β1 to receptor, was also highly upregulated in LysM-cre/Tlr4flox/floxmouse kidney after UUO. Thus, macrophages lacking TLR4 showed an anti-inflammatory and anti-fibrotic phenotype as demonstrated by high levels of Bambi but low levels of TGF-b/Smad3 signaling, MMT, and renal fibrosis in vivoand in vitro. Deletion of macrophage-specific TLR4 ameliorates obstructive renal injury by promoting anti-inflammatory macrophage polarization and inhibiting MMT-mediated renal fibrosis via a Bambi-dependent mechanism. Results from this study suggest that targeting the macrophage-specific TLR4 pathway may represent a novel therapeutic intervention for renal inflammation and fibrosis.

Transcript levels for extracellular matrix proteins are altered in MK5-deficient cardiac ventricular fibroblasts

MK5 is a protein serine/threonine kinase activated by p38 MAPK and the atypical MAPKs ERK3 and ERK4. Although little is known of the physiological role of MK5 in the heart, both hypertrophic growth and the increase in collagen 1-α1 mRNA induced by increased afterload are attenuated in hearts of MK5 haploinsufficient (MK5+/−) mice. MK5 transcripts are detected at high levels in the left ventricular myocardium; however, MK5 immunoreactivity is detected in adult cardiac fibroblasts, but not myocytes. The present study was to determine if MK5 has a potential role in remodeling of the extracellular matrix. Ventricular fibroblasts were isolated from MK5+/+, MK5+/−, or MK5−/− mice and maintained in culture on either compliant (8 kPa) or rigid substrates to obtain quiescent fibroblasts or activated myofibroblasts, respectively. In quiescent fibroblasts, reduced MK5 had little effect: BMP7 and TGF-β1 mRNA was increased in MK5+/− and MK5−/−.cells, respectively. Ang-II altered the abundance of numerous transcripts in an MK5-sensitive manner. Both collagen 1-α1 mRNA and secreted type 1 collagen immunoreactivity were increased by Ang-II in wild type but not MK5-deficient fibroblasts. The effects of deleting MK5 were quite different in myofibroblasts: both the abundance of collagen 1-α1 mRNA and secreted type 1 collagen immunoreactivity elevated in the absence of added Ang-II and addition of Ang-II failed to evoke a further increase in either. In addition, whereas type I collagen immunoreactivity was distributed throughout the cytosol of wild-type myofibroblasts, it was perinuclear in MK5−/− myofibroblasts. Furthermore, in MK5-deficient myofibroblasts the abundance of collagen 3-α2, Timp3, Smad 6, Smad 7, TGF-β3, and snail homolog 1 transcripts was increased whereas integrin β3, latent TGF-β binding protein 1, thrombospondin 1, hepatocyte growth factor, and interleukin 13 were decreased. Finally, fibroblast contraction was decreased upon knocking down MK5. These results indicate that MK5 may be involved in fibroblast-mediated regulation of extracellular matrix homeostasis.

Proteomic discovery of substrates of the cardiovascular protease ADAMTS7

The protease ADAMTS7 functions in the extracellular matrix (ECM) of the cardiovascular system. However, its physiological substrate specificity and mechanism of regulation remain to be explored. To address this, we conducted an unbiased substrate analysis using terminal amine isotopic labeling of substrates (TAILS). The analysis identified candidate substrates of ADAMTS7 in the human fibroblast secretome, including proteins with a wide range of functions, such as collagenous and noncollagenous extracellular matrix proteins, growth factors, proteases, and cell-surface receptors. It also suggested that autolysis occurs at Glu-729–Val-730 and Glu-732–Ala-733 in the ADAMTS7 Spacer domain, which was corroborated by N-terminal sequencing and Western blotting. Importantly, TAILS also identified proteolysis of the latent TGF-β–binding proteins 3 and 4 (LTBP3/4) at a Glu-Val and Glu-Ala site, respectively. Using purified enzyme and substrate, we confirmed ADAMTS7-catalyzed proteolysis of recombinant LTBP4. Moreover, we identified multiple additional scissile bonds in an N-terminal linker region of LTBP4 that connects fibulin-5/tropoelastin and fibrillin-1–binding regions, which have an important role in elastogenesis. ADAMTS7-mediated cleavage of LTBP4 was efficiently inhibited by the metalloprotease inhibitor TIMP-4, but not by TIMP-1 and less efficiently by TIMP-2 and TIMP-3. As TIMP-4 expression is prevalent in cardiovascular tissues, we propose that TIMP-4 represents the primary endogenous ADAMTS7 inhibitor. In summary, our findings reveal LTBP4 as an ADAMTS7 substrate, whose cleavage may potentially impact elastogenesis in the cardiovascular system. We also identify TIMP-4 as a likely physiological ADAMTS7 inhibitor.

Reversion of in vivo fibrogenesis by novel chromone scaffolds.

BackgroundMyofibroblasts are known to play a key role in the development of idiopathic pulmonary fibrosis (IPF). Two drugs, pirfenidone and nintedanib, are the only approved therapeutic options for IPF, but their applications are limited due to their side effects. Thus, curative IPF drugs represent a huge unmet medical need.MethodsA mouse hepatic stellate cell (HSC) line was established that could robustly differentiate into myofibroblasts upon treatment with TGF-β. Eupatilin was assessed in diseased human lung fibroblasts from IPF patients (DHLFs) as well as in human lung epithelial cells (HLECs). The drug's performance was extensively tested in a bleomycin-induced lung fibrosis model (BLM). Global gene expression studies and proteome analysis were performed.FindingsEupatilin attenuated disease severity of BLM in both preventative and therapeutic studies. The drug inhibited the in vitro transdifferantiation of DHLFs to myofibroblasts upon stimulation with TGF-β. No such induction of the in vitro transdifferantiation was observed in TGF-β treated HLECs. Specific carbons of eupatilin were essential for its anti-fibrotic activity. Eupatilin was capable of dismantling latent TGF-β complex, specifically by eliminating expression of the latent TGF-β binding protein 1 (LTBP1), in ECM upon actin depolymerization. Unlike eupatilin, pirfenidone was unable to block fibrosis of DHLFs or HSCs stimulated with TGF-β. Eupatilin attenuated phosphorylation of Smad3 by TGF-β. Eupatilin induced myofibroblasts to dedifferentiate into intermediate HCS-like cells.InterpretationEupatilin may act directly on pathogenic myofibroblasts, disarming them, whereas the anti-fibrotic effect of pirfenidone may be indirect. Eupatilin could increase the efficacy of IPF treatment to curative levels.

Fabrication of Nanofibrous PVA/Alginate-Sulfate Substrates for Growth Factor Delivery.

Providing affinity sites on alginate (ALG) matrix enables specific binding of growth factors to the polymer backbone and allows their release in a controlled fashion. In this study, we used a blend of alginate sulfate (ALG-S) and polyvinyl alcohol (PVA) to fabricate electrospun scaffolds capable of delivering a heparin-like growth factor, transforming growth factor-beta1 (TGF-β1). The alginate was sulfated with different degrees of sulfation (DS, from 0.8, 3.4 to 12.4%) by a simple process. The success of sulfation was determined by Fourier-transform infrared spectroscopy (FTIR), nuclear magnetic resonance spectroscopy (NMR), elemental analysis, ultraviolet (UV) spectroscopy and staining with dimethylmethylene blue. The physical-mechanical properties of nanofibrous mats were characterized by scanning electron microscopy (SEM), FTIR, energy-dispersive X-ray spectroscopy (EDX), tensile strength and mass loss analysis. Additionally, the release kinetics of transforming growth factor-β1 (TGF-β1) from PVA/ALG-S and PVA/ALG scaffolds were compared. The results showed that the binding and entrapment of TGF-β1 to the nanofibrous scaffolds are improved by the addition of sulfate group to alginate. In conclusion, our results support that nanofibrous scaffold based on PVA/ALG-S can deliver growth factors in tissue engineering application. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 403-413, 2019.

Impairment of chondrogenesis and microfibrillar network in Adamtsl2 deficiency.

Mutations in the a disintegrin and metalloproteinase with thrombospondin motif-like 2 ( ADAMTSL2) gene are responsible for the autosomal recessive form of geleophysic dysplasia, which is characterized by short stature, short extremities, and skeletal abnormalities. However, the exact function of ADAMTSL2 is unknown. To elucidate the role of this protein in skeletal development, we generated complementary knockout (KO) mouse models with either total or chondrocyte Adamtsl2 deficiency. We observed that the Adamtsl2 KO mice displayed skeletal abnormalities reminiscent of the human phenotype. Adamtsl2 deletion affected the growth plate formation with abnormal differentiation and proliferation of chondrocytes. In addition, a TGF-β signaling impairment in limbs lacking Adamtsl2 was demonstrated. Further investigations revealed that Adamtsl2 KO chondrocytes failed to establish a microfibrillar network composed by fibrillin1 and latent TGF-β binding protein 1 fibrils. Chondrocyte Adamtsl2 KO mice also exhibited dwarfism. These studies uncover the function of Adamtsl2 in the maintenance of the growth plate ECM by modulating the microfibrillar network.-Delhon, L., Mahaut, C., Goudin, N., Gaudas, E., Piquand, K., Le Goff, W., Cormier-Daire, V., Le Goff, C. Impairment of chondrogenesis and microfibrillar network in Adamtsl2 deficiency.

Abstract IA08: Engineering T cells to resist and convert immunosuppressive tumor microenvironments

Abstract A major challenge in adoptive T-cell therapy for solid tumors is the harsh immunosuppressive tumor microenvironment. Soluble factors such as transforming growth factor beta (TGF-β) play key roles in tumor immune evasion. Here, we report methods by which CARs can be engineered to respond to soluble antigens, and describe a novel TGF-β binding chimeric antigen receptor that rewires the T-cell response to TGF-β, converting this potent immunosuppressive molecule into a stimulant. We additionally elucidate mechanistic details of how CARs can be triggered by soluble ligands, as well as methods by which the signaling strength and ligand sensitivity of such CARs can be tuned. Given the modular nature of the engineered receptor, this work informs future efforts to redirect T-cell responses to extracellular soluble molecules. Citation Format: ZeNan L. Chang, Michael H. Lorenzini, Yvonne Y. Chen. Engineering T cells to resist and convert immunosuppressive tumor microenvironments [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr IA08.

Octreotide ameliorates dermal fibrosis in bleomycin-induced scleroderma

Insulin-like growth factor (IGF)-I is a differentiation and growth factor. Antifibrotic action of octreotide has been reported in pulmonary fibrosis. The present study aimed to research the prophylactic and therapeutic potential of octreotide on a bleomycin (BLM)-induced experimental scleroderma model. Sixty Balb/c female mice were divided into 6 groups. Daily subcutaneous BLM (100 μg) was injected for 3 weeks in groups II and III and for 6 weeks in groups V and VI. Octreotide (100 μg/kg per day) was injected subcutaneously for the first 3 weeks in group III (prophylactic) and the second 3 weeks in group VI (therapeutic). Mice in groups I, II, and III were sacrificed at the end of the third week, while mice in groups IV, V, and VI were sacrificed at the end of the sixth week. Repeated BLM applications increased dermal inflammatory cell counts and dermal thickness, and led to dermal fibrosis at both the third and sixth weeks. Moreover, mRNA expressions of TGF-β1 and IGF binding protein (IGFBP)-3 and -5 were higher in the BLM- injected sham groups. On the other hand, IGFBP-3 and -5 mRNA expressions were significantly decreased in both the prophylactic and therapeutic octreotide groups. Similarly, octreotide decreased dermal inflammatory infiltrations and dermal thickness. Octreotide has antifibrotic actions on experimentally induced dermal fibrosis. It can be suggested that IGF-I plays pathogenic roles, and octreotide is a candidate for research in the treatment of scleroderma.

Utility of the Teslar Facial Massager for Skin Elasticity and the Mechanism of its Effects

The Teslar is a facial massager that emits a weak electric current, where users have reported a beneficial effect on skin elasticity with continued use. Accordingly, we conducted a clinical utility study and a comprehensive gene analysis, with cultured human fibroblasts to investigate the utility and mechanism of this treatment. In this clinical utility study, we found significant improvement in skin elasticity in Teslar treatments, compared to controls after two weeks of treatment. In cell experiments, we found that adenosine triphosphate synthesis and collagen contraction were promoted in fibroblasts cultured in type I collagen gel, following Teslar treatment. We considered that Teslar treatment exerted a structurally regenerative effect on the dermal matrix, based on the results of GeneChip® Expression Analysis. In particular, we demonstrated that Teslar treatment promotes type I collagen mRNA expression and fibulin-5/DANCE (Developmental arteries and neural crest EGF (epidermal growth factor)-like) mRNA expression and protein levels, which are reduced with aging. We also found increases in LTBP-3 (Latent TGF-β binding protein-3) and CSPG4 (Chondroitin sulfate proteoglycan 4) mRNA expression levels. Based on these results, we considered that Teslar treatment promoted dermal regeneration and recovery of skin elasticity.

Preparation and evaluation of anti-renal fibrosis activity of novel truncated TGF-β receptor type II.

Production of excessive transforming growth factor-beta 1 (TGF-β1) with elevated TGF-β1 activity has been implicated in renal fibrosis via renal epithelial cells activation and collagen deposition. As such, attenuating the binding of TGF-β1 to its receptor TGF-beta receptor type II (TGF-βRII) in TGF-β1-dependent signaling is an attractive target for the control of renal fibrosis. Here, we verified the interaction between novel truncated human TGF-βRII (thTβRII, Thr23-Gln166) and TGF-β1, prepared thTβRII in Escherichia coli, and assessed the effects of thTβRII on TGF-β1-induced human kidney epithelial cells (HK-2) and unilateral ureteral obstruction (UUO) model of renal fibrosis. Our data showed that thTβRII accounted for up to 20% of the total protein and 40% of the inclusion bodies of whole cell lysates under the optimal conditions (0.8 mM IPTG and 25°C for 6 H). Most of the expressed protein in inclusion body was refolded by dialysis refolding procedures and purified by Ni2+ -IDA affinity chromatography. Furthermore, thTβRII decreased type I collagen and α-smooth muscle actin protein expression in TGF-β1-induced HK-2 cells, and ameliorated kidney morphology and fibrotic responses in fibrosis animal. These findings indicate that thTβRII holds great promise for developing new treatments for renal fibrosis.