Presence Of TGF-β1 Research Articles

Cementoblastic lineage formation in the cross-talk between stem cells of human exfoliated deciduous teeth and epithelial rests of Malassez cells.

The purpose of this study was to evaluate the synergistic effect of epithelial rests of Malassez cells (ERM) and transforming growth factor-β1 (TGF-β1) on proliferation, cementogenic and osteogenic differentiation of stem cells derived from human exfoliated deciduous teeth (SHED). SHED were co-cultured with ERM with/without TGF-β1. Then, SHED proliferation, morphological appearance, alkaline phosphatase (ALP) activity, mineralization behaviour and gene/protein expression of cemento/osteoblastic phenotype were evaluated. TGF-β1 enhanced SHED proliferation when either cultured alone or co-cultured with ERM. ERM induced the cementoblastic differentiation of SHED which was significantly accelerated when treated with TGF-β1. This activity was demonstrated by high ALP activity, strong mineral deposition and upregulation of cementum/bone-related gene and protein expressions (i.e. ALP, collagen type I, bone sialoprotein, osteocalcin and cementum attachment protein). ERM were able to induce SHED differentiation along the cemento/osteoblastic lineage that was triggered in the presence of TGF-β1. The cemento/osteoblastic differentiation capability of SHED possesses a therapeutic potential in endodontic and periodontal tissue engineering.

Abstract 4125: SSA, a novel sulindac derivative, inhibits breast cancer cell invasion and migration

Abstract Nonsteroidal anti-inflammatory drugs (NSAIDs), such as sulindac, have been reported for striking chemopreventive activity in various types of human cancer, including breast cancer. However, few studies have reported that sulindac is able to prevent tumor metastasis. In this study, we aim to study anti-metastatic activity of sulindac in breast cancer and explore the mechanistic basis responsible for this activity, which we hypothesize may involve a cyclooxygenase (COX) independent mechanism. Numerous studies have reported that the anticancer properties of sulindac might involve, at least partially, a non-COX mediated mechanism, which provides an insight into development of safer and more efficacious new derivatives. We have previously described a chemical derivative of sulindac, referred to as sulindac sulfide amide (SSA), which lacks inhibitory effect on COX-1 or -2, yet shows greater potency to inhibit tumor cell growth compared with sulindac sulfide (SS). Our results showed that SSA treatment at a sub-cytotoxic concentration (4μM) for 36 h significantly inhibits both migration and invasion of highly aggressive MDA-MB-231, BT-20 and SK-BR-3 breast tumor cells. When treating non-invasive breast MCF-7 cells with TGF-β1 to induce their motility, we found that SSA could significantly counteract the induced motility of these cells. These results indicate that SSA can suppress breast tumor cell invasion by modulation of TGF-β1 signaling. We further studied Smad-2/3 that are important downstream components in the TGF-β1 signaling pathway and found that SSA could inhibit Smad-2/3 phosphorylation and reduce their translocation to the nucleus. In addition, Smad-2/3 knockdown could mimic the anti-invasive effect of SSA in MDA-MB-231 cells, regardless of the presence of TGF-β1. In summary, we demonstrate that the non-COX inhibitory derivative, SSA can suppress breast cancer cell invasion and migration. These observations suggest that inhibition of TGF-β1/Smad signaling is responsible for the inhibitory effect of SSA on breast tumor cell invasion and migration. Citation Format: Bin Yi, Xingling Feng, Ruixia Ma, Xiaoguo Zhang, Hong Chang, Hongyou Zhao, Ziping Liang, Xi Chen, Xiuhua Hu, Gary Piazza, Yaguang Xi. SSA, a novel sulindac derivative, inhibits breast cancer cell invasion and migration. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4125. doi:10.1158/1538-7445.AM2015-4125

LXA4 actions direct fibroblast function and wound closure

Timely resolution of inflammation is crucial for normal wound healing. Resolution of inflammation is an active biological process regulated by specialized lipid mediators including the lipoxins and resolvins. Failure of resolution activity has a major negative impact on wound healing in chronic inflammatory diseases that is manifest as excess fibrosis and scarring. Lipoxins, including Lipoxin A4 (LXA4), have known anti-fibrotic and anti-scarring properties. The goal of this study was to elucidate the impact of LXA4 on fibroblast function. Mouse fibroblasts (3T3 Mus musculus Swiss) were cultured for 72 h in the presence of TGF-β1, to induce fibroblast activation. The impact of exogenous TGF-β1 (1 ng/mL) on LXA4 receptor expression (ALX/FPR2) was determined by flow cytometry. Fibroblast proliferation was measured by bromodeoxyuridine (BrdU) labeling and migration in a “scratch” assay wound model. Expression of α-smooth muscle actin (α-SMA), and collagen types I and III were measured by Western blot. We observed that TGF-β1 up-regulates LXA4 receptor expression, enhances fibroblast proliferation, migration and scratch wound closure. α-SMA levels and Collagen type I and III deposition were also enhanced. LXA4 slowed fibroblast migration and scratch wound closure at early time points (24 h), but wound closure was equal to TGF-β1 alone at 48 and 72 h. LXA4 tended to slow fibroblast proliferation at both concentrations, but had no impact on α-SMA or collagen production by TGF-β1 stimulated fibroblasts. The generalizability of the actions of resolution molecules was examined in experiments repeated with resolvin D2 (RvD2) as the agonist. The activity of RvD2 mimicked the actions of LXA4 in all assays, through an as yet unidentified receptor. The results suggest that mediators of resolution of inflammation enhance wound healing and limit fibrosis in part by modulating fibroblast function.

Abstract 367: Lysyl Oxidase Expression by Cardiac Fibroblasts is Regulated by Integrin-mediated Signaling

Cardiac fibrosis following myocardial infarction (MI) leads to reduced cardiac function, and contributes to heart failure and mortality. Recent studies shown the extent of adverse remodeling may be mitigated by therapeutic strategies which regulate cardiac fibroblast mediated-remodeling. Since cross-linking by lysyl oxidase (LOX) increases following MI and alters the mechanical properties of the infarct, it is critical to characterize how its expression is regulated by CFs post-MI. While LOX expression is attributable to TGF-β1 signaling, we hypothesize that changes in the stiffness and composition of the ECM can also alter LOX expression via integrin-mediated signaling. To investigate this, we isolated CFs from healthy left ventricle (LV) and infarcted cardiac fibroblasts (ICFs) from 1 week post-MI LV and cultured them on tissue culture plastic (TCP) and collagen I-coated plates (COL) in serum-free media for 48 hours to assess the expression of genes associated with LOX signaling, fibrosis, and myofibroblast activation. Our results show an upregulation of LOX gene expression in both CFs and ICFs when cultured on COL and this is further emphasized with the presence of TGF-β1 (Fig. 1A). Gene expression of col1α1, integrin β1 subunit and αSMA (Fig. 1B-D) also exhibit similar upregulation. Ongoing studies will investigate how altered substrate stiffness and composition affect gene expression of LOX and other genes associated with fibrosis. By understanding the effect of the physical microenvironment on the expression of fibrotic genes including LOX, we aim to develop novel therapeutic strategies to attenuate cardiac fibrosis and thus improve cardiac recovery following MI.

Chondrogenically primed mesenchymal stem cell-seeded alginate hydrogels promote early bone formation in critically-sized defects

Hypertrophic cartilaginous grafts can be engineered in vitro using bone marrow derived Mesenchymal Stem Cells (MSCs). When such engineered tissues are implanted in vivo they have been shown to induce bone formation by recapitulating aspects of the developmental process of endochondral ossification. Alginate, a naturally sourced and biocompatible hydrogel, offers an attractive 3D environment to facilitate the in vitro chondrogenesis of MSCs. Furthermore, such alginate hydrogels can potentially be used to engineer cartilage tissues of scale to promote endochondral bone regeneration in large bone defects. The aim of this study was to investigate the ability of chondrogenically-primed MSC-laden alginate hydrogels to induce healing in two distinct critically-sized defect models. Bone marrow derived MSCs were seeded into alginate hydrogels, chondrogenically primed in vitro in the presence of TGF-β3 and then implanted into either a critically-sized rat cranial or femoral defect. μCT analysis 4weeks post-implantation revealed significantly higher levels of mineralization within the femoral defects treated with MSC-laden alginate hydrogels compared to untreated empty controls, with similar results observed within the cranial defects. However, any newly deposited bone was generated appositional to the alginate material, and occurred only superficially or where the alginate was seen to degrade. Alginate material was found to persist within both orthotopic locations 8weeks post-implantation, with its slow rate of degradation appearing to prevent complete bone regeneration. In conclusion, while chondrogenically primed MSC–alginate constructs can act as templates to treat critically-sized defects within bones formed through either intramembranous or endochondral ossification, further optimization of the degradation kinetics of the hydrogel itself will be required to accelerate bone tissue deposition and facilitate complete regeneration of such defects.

Increased stability of heterogeneous ribonucleoproteins by a deacetylase inhibitor

Splicing factors are often influenced by various signaling pathways, contributing to the dynamic changes of protein isoforms in cells. Heterogeneous ribonucleoproteins (hnRNPs) regulate many steps of RNA metabolism including pre-mRNA splicing but their control by cell signaling particularly through acetylation and ubiquitination pathways remains largely unknown. Here we show that TSA, a deacetylase inhibitor, reduced the ratio of Bcl-x splice variants Bcl-xL/xS in MDA-MB-231 breast cancer cells. This TSA effect was independent of TGFβ1; however, only in the presence of TGFβ1 was TSA able to change the splicing regulators hnRNP F/H by slightly reducing their mRNA transcripts but strongly preventing protein degradation. The latter was also efficiently prevented by lactacystin, a proteasome inhibitor, suggesting their protein stability control by both acetylation and ubiquitination pathways. Three lysines K87, K98 and K224 of hnRNP F are potential targets of the mutually exclusive acetylation or ubiquitination (KAc/Ub) in the protein modification database PhosphoSitePlus. Mutating each of them but not a control non-KAc/Ub (K68) specifically abolished the TSA enhancement of protein stability. Moreover, mutating K98 (K98R) and K224 (K224R) also abolished the TSA regulation of alternative splicing of a Bcl-x mini-gene. Furthermore, about 86% (30 of 35) of the multi-functional hnRNP proteins in the database contain lysines that are potential sites for acetylation/ubiquitination. We demonstrate that the degradation of three of them (A1, I and L) are also prevented by TSA. Thus, the deacetylase inhibitor TSA enhances hnRNP F stability through the KAc/Ub lysines, with some of them essential for its regulation of alternative splicing. Such a regulation of protein stability is perhaps common for a group of hnRNPs and RNA metabolism.

HDAC inhibitors induce epithelial-mesenchymal transition in colon carcinoma cells.

The effects of histone deacetylase (HDAC) inhibitors on epithelial-mesenchymal transition (EMT) differ in various types of cancers. We investigated the EMT phenotype in four colon cancer cell lines when challenged with HDAC inhibitors trichostatin A (TSA) and valproic acid (VPA) with or without transforming growth factor-β1 (TGF-β1) treatment. Four colon cancer cell lines with different phenotypes in regards to tumorigenicity, microsatellite stability and DNA mutation were used. EMT phenotypes were assessed by the expression of E-cadherin and vimentin using western blot analysis, immunofluorescence, quantitative real-time RT-PCR following treatment with TSA (100 or 200 nM) or VPA (0.5 mM) with or without TGF-β1 (5 ng/ml) for 24 h. Biological EMT phenotypes were also evaluated by cell morphology, migration and invasion assays. TSA or VPA induced mesenchymal features in the colon carcinoma cells by a decrease in E-cadherin and an increase in vimentin expression at the mRNA and protein levels. Confocal microscopy revealed membranous attenuation or nuclear translocation of E-cadherin and enhanced expression of vimentin. These responses occurred after 6 h and increased until 24 h. Colon cancer cells changed from a round or rectangular shape to a spindle shape with increased migration and invasion ability following TSA or VPA treatment. The susceptibility to EMT changes induced by TSA or VPA was comparable in microsatellite stable (SW480 and HT29) and microsatellite unstable cells (DLD1 and HCT116). TSA or VPA induced a mesenchymal phenotype in the colon carcinoma cells and these effects were augmented in the presence of TGF-β1. HDAC inhibitors require careful caution before their application as new anticancer drugs for colon cancers.

Inflammatory microenvironment and tumor necrosis factor alpha as modulators of periostin and CCN2 expression in human non-healing skin wounds and dermal fibroblasts

Non-healing skin wounds remain a significant clinical burden, and in recent years, the regulatory role of matricellular proteins in skin healing has received significant attention. Periostin and CCN2 are both upregulated at day 3 post-wounding in murine skin, where they regulate aspects of the proliferative phase of repair including mesenchymal cell infiltration and myofibroblast differentiation. In this study, we examined 1) the wound phenotype and expression patterns of periostin and CCN2 in non-healing skin wounds in humans and 2) the regulation of their expression in wound fibroblasts by tumor necrosis factor α (TNFα) and transforming growth factor-β1 (TGF-β1). Chronic skin wounds had a pro-inflammatory phenotype, characterized by macrophage infiltration, TNFα immunoreactivity, and neutrophil infiltration. Periostin, but not CCN2, was significantly suppressed in non-healing wound edge tissue at the mRNA and protein level compared with non-involved skin. In vitro, human wound edge fibroblasts populations were still able to proliferate and contract collagen gels. Compared to cells from non-involved skin, periostin and α-SMA mRNA levels increased significantly in the presence of TGF-β1 in wound cells and were significantly decreased by TNFα, but not those of Col1A2 or CCN2. In the presence of both TGF-β1 and TNFα, periostin and α-SMA mRNA levels were significantly reduced compared to TGF-β1 treated wound cells. Effects of TGF-β1 and TNFα on gene expression were also more pronounced in wound edge cells compared to non-involved fibroblasts. We conclude that variations in the expression of periostin and CCN2, are related to an inflammatory microenvironment and the presence of TNFα in human chronic wounds.

NURSING SIG – ORAL PRESENTATIONS

Aim: In patients with idiopathic pulmonary fibrosis (IPF), a fatal interstitial lung disease, advancing fibrosis, driven by transforming growth factor-beta 1 (TGFβ1), results in excessive extracellular matrix (ECM) deposition. Our recent studies have shown that the glycoprotein fibulin-1 is increased in serum and lung tissue from patients with IPF and that their fibroblasts, which are the main effector cells for fibrosis, produce more fibulin-1 than those from healthy people. What regulates fibroblast derived fibulin-1 in people with IPF is unknown. Methods: Primary parenchymal fibroblasts were derived from five patients with IPF and five patients without IPF (Non-IPF). The production of fibulin-1 in the presence of cigarette smoke extract (CSE), foetal bovine serum (FBS) or transforming growth factor (TGF)β1 in the presence or absence of BIBF1120 (a triple kinase inhibitor) was measured by RT PCR or ELISA. Cytotoxicity of BIBF1120 was investigated by LDH-assay. Proliferation was measured by MTT and CyQuant and cell attachment was assessed by toluidine blue absorbance in fibroblasts from both groups. Results: CSE did not modulate fibulin-1 expression. Fibroblasts from IPF patients proliferated faster in 10% FBS and deposited more fibulin-1 in the presence of TGF-β1, compared to Non-IPF fibroblasts. Attachment did not differ between the fibroblast groups. BIBF1120 reduced proliferation and FBLN-1 deposition in the ECM, but did not influence attachment of fibroblasts. There were no differences in the responses between fibroblasts from patients with and without IPF. Conclusion: BIBF1120 inhibits fibroblastic processes that contribute to the pathogenesis of IPF. This new therapeutic may be effective in targeting the development of fibrosis in IPF patients.

Evaluation of Cartilage Repair by Mesenchymal Stem Cells Seeded on a PEOT/PBT Scaffold in an Osteochondral Defect.

The main objective of this study was to evaluate the effectiveness of a mesenchymal stem cell (MSC)-seeded polyethylene-oxide-terephthalate/polybutylene-terephthalate (PEOT/PBT) scaffold for cartilage tissue repair in an osteochondral defect using a rabbit model. Material characterisation using scanning electron microscopy indicated that the scaffold had a 3D architecture characteristic of the additive manufacturing fabrication method, with a strut diameter of 296±52μm and a pore size of 512±22μm×476±25μm×180±30μm. In vitro optimisation revealed that the scaffold did not generate an adverse cell response, optimal cell loading conditions were achieved using 50μg/ml fibronectin and a cell seeding density of 25×10(6)cells/ml and glycosaminoglycan (GAG) accumulation after 28days culture in the presence of TGFβ3 indicated positive chondrogenesis. Cell-seeded scaffolds were implanted in osteochondral defects for 12weeks, with cell-free scaffolds and empty defects employed as controls. On examination of toluidine blue staining for chondrogenesis and GAG accumulation, both the empty defect and the cell-seeded scaffold appeared to promote repair. However, the empty defect and the cell-free scaffold stained positive for collagen type I or fibrocartilage, while the cell-seeded scaffold stained positive for collagen type II indicative of hyaline cartilage and was statistically better than the cell-free scaffold in the blinded histological evaluation. In summary, MSCs in combination with a 3D PEOT/PBT scaffold created a reparative environment for cartilage repair.

L-thyroxine promotes a proliferative airway smooth muscle phenotype in the presence of TGF-β1.

Hypothyroidism may reduce, whereas hyperthyroidism may aggravate, asthma symptoms. The mechanisms underlying this relationship are largely unknown. Since thyroid hormones have central roles in cell growth and differentiation, we hypothesized that airway remodeling, in particular increased airway smooth muscle (ASM) mass, may be involved. To address this hypothesis, we investigated the effects of triiodothyronine (T3) and l-thyroxine (T4) in the absence and presence of the profibrotic transforming growth factor (TGF)-β1 on human ASM cell phenotype switching. T3 (1-100 nM) and T4 (1-100 nM) did not affect basal ASM proliferation. However, when combined with TGF-β1 (2 ng/ml), T4 synergistically increased the proliferative response, whereas only a minor effect was observed for T3. In line with a switch from a contractile to a proliferative ASM phenotype, T4 reduced the TGF-β1-induced contractile protein expression by ∼50%. Cotreatment with T3 reduced TGF-β1-induced contractile protein expression by ∼25%. The synergistic increase in proliferation was almost fully inhibited by the integrin αvβ3 antagonist tetrac (100 nM), whereas no significant effects of the thyroid receptor antagonist 1-850 (3 μM) were observed. Inhibition of MEK1/2, downstream of the integrin αvβ3, also inhibited the T4- and TGF-β1-induced proliferative responses. Collectively, the results indicate that T4, and to a lesser extent T3, promotes a proliferative ASM phenotype in the presence of TGF-β1, which is predominantly mediated by the membrane-bound T4 receptor αvβ3. These results indicate that thyroid hormones may enhance ASM remodeling in asthma, which could be of relevance for hyperthyroid patients with this disease.

Substrate stiffness together with soluble factors affects chondrocyte mechanoresponses.

Tissue cells sense and respond to differences in substrate stiffness. In chondrocytes, it has been shown that substrate stiffness regulates cell spreading, proliferation, chondrogenic gene expression, and TGF-β signaling. But how the substrate stiffness together with soluble factors influences the mechanical properties of chondrocyte is still unclear. In this study, we cultured goat articular chondrocytes on polyacrylamide gels of 1, 11, and 90 kPa (Young's modulus), and measured cellular stiffness, traction force, and response to stretch in the presence of TGF-β1 or IL-1β. We found that TGF-β1 increased cellular stiffness and traction force and enhanced the response to stretch, while IL-1β increased cellular stiffness, but lowered traction force and weakened the response to stretch. Importantly, the effects of TGF-β1 on chondrocyte mechanics were potent in cells cultured on 90 kPa substrates, while the effects of IL-1β were potent on 1 kPa substrates. We also demonstrated that such changes of chondrocyte mechanoresponse were due to not only the changes of actin cytoskeleton and focal adhesion, but also the alteration of chondrocyte extracellular matrix synthesis. Taken together, these results provide insights into how chondrocytes integrate physical and biochemical cues to regulate their biomechanical behavior, and thus have implications for the design of optimized mechanical and biochemical microenvironments for engineered cartilage.

NFkappaB activation is essential for miR-21 induction by TGFβ1 in high glucose conditions

Transforming growth factor beta1 (TGFβ1) is a pleiotropic growth factor with a very broad spectrum of effects on wound healing. Chronic non-healing wounds such as diabetic foot ulcers express reduced levels of TGFβ1. On the other hand, our previous studies have shown that the microRNA miR-21 is differentially regulated in diabetic wounds and that it promotes migration of fibroblast cells. Although interplay between TGFβ1 and miR-21 are studied in relation to cancer, their interaction in the context of chronic wounds has not yet been investigated. In this study, we examined if TGFβ1 could stimulate miR-21 in fibroblasts that are subjected to high glucose environment. MiR-21 was, in fact, induced by TGFβ1 in high glucose conditions. The induction by TGFβ1 was dependent on NFκB activation and subsequent ROS generation. TGFβ1 was instrumental in degrading the NFκB inhibitor IκBα and facilitating the nuclear translocation of NFκB p65 subunit. EMSA studies showed enhanced DNA binding activity of NFκB in the presence of TGFβ1. ChIP assay revealed binding of p65 to miR-21 promoter. NFκB activation was also required for the nuclear translocation of Smad 4 protein and subsequent direct interaction of Smad proteins with primary miR-21 as revealed by RNA-IP studies. Our results show that manipulation of TGFβ1–NFκB–miR-21 pathway could serve as an innovative approach towards therapeutics to heal diabetic ulcers.

Effects of different transforming growth factor beta (TGF-β) isomers on wound closure of bone cell monolayers

This study aimed at determining the role of the transforming growth factor-beta (TGF-β) isomers and their combinations in bone cell behaviour using MG63 cells. The work examined how TGF-β1, 2 and 3 and their solvent and carrier (HCl and BSA, respectively) effected cell morphology, cell proliferation and integrin expression. This study also aimed at examining how the TGF-βs and their solvent and carrier influenced wound closure in an in vitro wound closure model and how TGF-βs influence extracellular matrix (ECM) secretion and integrin expression. The wound healing response in terms of healing rate to the TGF-βs and their solvent/carrier was investigated in 300μm±10–30μm SD wide model wounds induced in fully confluent monolayers of MG63 bone cells. The effect of different TGF-β isomers and their combinations on proliferation rate and cell length of human bone cells were also assessed. Immunostaining was used to determine if TGF-βs modifies integrin expression and ECM secretion by the bone cells. Imaging with WSPR allowed observation of the focal contacts without the need for immunostaining. The wound healing results indicated that TGF-β3 has a significant effect on the wound healing process and its healing rate was found to be higher than the control (p<0.001), TGF-β1 (p<0.001), TGF-β2 (p<0.001), BSA/HCl (p<0.001) and HCl (p<0.001) in ascending order. It was also found that TGF-β1 and TGF-β2 treatment significantly improved wound closure rate in comparison to the controls (p<0.001). All TGF-β combinations induced a faster healing rate than the control (p<0.001). It was expected that the healing rate following treatment with TGF-β combinations would be greater than those healing rates following treatments with TGF-β isomers alone, but this was not the case. The results also suggest that cell morphological changes were observed significantly more in cells treated with TGF-β(2+3) and TGF-β(1+3) (p<0.001). Any cell treated with TGF-β1, TGF-β(1+2) and TGF-β(1+2+3) showed significantly less elongation compared to the control and other TGF-β isomers. In terms of proliferation rate, TGF-β3 and TGF-β(2+3) increased cell numbers more than TGF-β1, TGF-β2 and other combinations. TGF-β1 and its combinations did not show significant proliferation and attachment compared to the control. Immunostaining indicated that treatment with TGF-β3 significantly enhanced the secretion of collagen type I, fibronectin and integrins α3 and β1. The WSPR experiments also indicated that TGF-βs influenced the distribution of focal contacts. In conclusion, combining TGF-β3 with any other TGF-β isomer resulted in a faster model wound closure rate (p<0.001), while treatment with TGF-β1 in any TGF-β combination reduced the healing rate (p<0.001). It can therefore be concluded that the presence of TGF-β1 has an inhibitory effect on bone wound healing while TGF-β3 had the opposite effect and increased the rate of wound closure in a 2 dimensional cell culture environment.

Therapeutic targeting of GSK3β enhances the Nrf2 antioxidant response and confers hepatic cytoprotection in hepatitis C

ObjectiveImpaired adaptive response to oxidative injuries is a fundamental mechanism central to the pathogenesis of chronic hepatitis C (CHC). Glycogen synthase kinase (GSK) 3β is an indispensable regulator of the...

TGF-β1 enhances T-cell PD-1 expression through a Smad3-dependant increase in transcription. (IRM4P.499)

Abstract Programmed cell death-1 (PD-1) is an inhibitory receptor highly expressed on T cells specific for viral and tumor antigens. TGF-β1 affects the tumor microenvironment and high serum levels are associated with both malignancy and pathogenesis in some chronic viral infections. However, the effect of TGF-β1 on PD-1 expression is unknown. PD-1 expression requires nuclear factor of activated T cells (NFATc1) activation through T cell receptor (TCR) signaling. We found that TGF-β1 signaling increases NFATc1-induced pdcd-1 mRNA and PD-1 expression on human peripheral CD4+ and CD8+ T cells. Smad2 and Smad3 are phosphorylated following TGF-β Receptor I binding and ultimately mediate gene transcription. We found that inhibition of TGFβRI kinase activity or Smad3 phosphorylation blocked TGF-β1-dependent PD-1 enhancement on T cells. Similarly, when compared to TCR stimulation alone, TGF-β1 increased PD-1 expression on murine wild-type (WT) but not on Smad3 knock-out (KO) CD4+ T cells. In contrast, Smad2 KO CD4+ T cells maintain increased PD-1 expression in the presence of TGF-β1, demonstrating a specific role for Smad3. We show that Smad3 increases pdcd-1 transcription by directly binding to a proximal region of the human PD-1 promoter and stabilizing NFATc1 binding. In addition to TGF-β1’s previously known effects on T cell function, our findings suggest that TGF-β1 mediates suppression via PD-1 upregulation. Smad3 may represent an additional target in therapeutic modulation of PD-1.

Fluvastatin-mediated suppression of IgE-induced cytokine production can be enhanced by TGFβ1 (HYP3P.403)

Abstract Fluvastatin, an HMG-CoA reductase inhibitor known for its role in the treatment of hypercholesterolemia and cardiovascular disease, has recently been shown to play a role in the immune response. Given the critical role that mast cells play in allergy and inflammatory diseases such as asthma, we assessed the effect of Fluvastatin on mast cell function. We also examined the impact of TGFβ1, a cytokine we have previously shown capable of limiting mast cell function, in combination with Fluvastatin. We find that Fluvastatin suppresses IgE-mediated cytokine production in mouse mast cells. Prior exposure to TGFβ1 enhanced the effects of Fluvastatin, such that the IC50 for Fluvastatin-mediated suppression was 3-4-fold lower in the presence of TGFβ1 than in its absence. Interestingly, this effect was found for IgE-induced production of IL-6 and TNF, while TGFβ1 did not alter the suppressive effects of Fluvastatin on IL-13 or MCP-1. Current experiments seek to unravel the mechanism behind this selective effect of TGFβ1.

Effect of platelet-rich plasma on chondrogenic differentiation in three-dimensional culture.

Platelet-rich plasma (PRP) may have the potential to enhance articular cartilage regeneration through release of growth factors including transforming growth factor isoforms. The purpose of this study was to investigate the potential for PRP to stimulate chondrogenic differentiation in three-dimensional PRP hydrogel constructs. Allogenic PRP was prepared using a double centrifugation protocol which resulted in a platelet concentration approximately 250% above baseline. Canine marrow stromal cells were encapsulated at 6.8×106 cells/ml in either 2% sodium alginate or in a 3:1 mixture of freshly prepared PRP and 2% alginate. PRP and alginate beads were cultured in chemically defined chondrogenic medium with and without 10 ng/ml TGF-β3. PRP cultures were additionally supplemented with frozen-thawed PRP. In the absence of TGF-β3, PRP had a mild stimulatory effect on cell proliferation. PRP did not stimulate cell proliferation in the presence of TGF-β3. Cells exposed to TGF-β3 accumulated significantly more GAG/DNA than those which were not, but there was not a statistically significant difference between alginate and PRP. Total collagen content was greater in PRP than in alginate, regardless of TGF-β3. Chondrogenesis in PRP was qualitatively and spatially different than that which occurred in conventional alginate beads and was characterized by isolated centers of intense chondrogenesis. Overall the results demonstrate that PRP alone weakly promotes chondroinduction of marrow stromal cells, and the effect is greatly augmented by TGF-β3.

Impact of Hydroxylase inhibitors on fibrosis associated with IBD (LB789)

Fibrosis is a major complication of inflammatory bowel disease (IBD) which cannot be adequately addressed with pharmacologic therapy. In IBD the treatment of fibrosis usually requires surgical resection of colonic segments and is responsible for 75% of surgical interventions in Crohn’s disease (CD). Pharmacological inhibition of hydroxylases ameliorates inflammation in multiple models of IBD. We hypothesized that hydroxylase inhibition may also impact fibrosis in IBD. To test this, the effect of the hydroxylase inhibitor DMOG was tested using an in vivo model of IBD. Mice given 2.5% dextran sodium sulphate (DSS) in drinking water for 5 days and allowed natural recovery for 14 days, developed inflammation and fibrosis, as shown by higher inflammation score, higher submucosal collagen‐I deposition and increased fibroblast infiltration through the colonic mucosa. Mice treated with intra peritoneal DMOG, exhibited less inflammation and lower collagen accumulation, as well as reduced fibroblast infiltration. To test whether this antifibrotic action was secondary to the anti‐inflammatory effect or due to a direct anti fibrotic action, an in vitro model was used. Fibroblasts were treated with TGF‐β1, the key mediator of healing and fibrosis. DMOG was given to the cells prior to TGF‐β1, and α‐smooth muscle actin (α‐SMA) and collagen‐I were analyzed as key fibrosis markers. Both markers were reduced by hydroxylase inhibitors in the presence of TGF‐β1, suggesting a direct antifibrotic action in fibroblasts. Furthermore, this effect was related to a reduced expression of Smad‐3 in human colonic fibroblast, a central effector of the TGF‐β pathway. In conclusion, hydroxylase inhibition reduces fibrosis in vitro and in vivo affecting, at least in part, the TGF‐β pathway. That could be a starting point to search for therapies that can ameliorate fibrosis in this and other diseases.Grant Funding Source: Supported by Science Foundation Ireland

Interleukin-1β Attenuates Myofibroblast Formation and Extracellular Matrix Production in Dermal and Lung Fibroblasts Exposed to Transforming Growth Factor-β1

One of the most potent pro-fibrotic cytokines is transforming growth factor (TGFβ). TGFβ is involved in the activation of fibroblasts into myofibroblasts, resulting in the hallmark of fibrosis: the pathological accumulation of collagen. Interleukin-1β (IL1β) can influence the severity of fibrosis, however much less is known about the direct effects on fibroblasts. Using lung and dermal fibroblasts, we have investigated the effects of IL1β, TGFβ1, and IL1β in combination with TGFβ1 on myofibroblast formation, collagen synthesis and collagen modification (including prolyl hydroxylase, lysyl hydroxylase and lysyl oxidase), and matrix metalloproteinases (MMPs). We found that IL1β alone has no obvious pro-fibrotic effect on fibroblasts. However, IL1β is able to inhibit the TGFβ1-induced myofibroblast formation as well as collagen synthesis. Glioma-associated oncogene homolog 1 (GLI1), the Hedgehog transcription factor that is involved in the transformation of fibroblasts into myofibroblasts is upregulated by TGFβ1. The addition of IL1β reduced the expression of GLI1 and thereby also indirectly inhibits myofibroblast formation. Other potentially anti-fibrotic effects of IL1β that were observed are the increased levels of MMP1, −2, −9 and −14 produced by fibroblasts exposed to TGFβ1/IL1β in comparison with fibroblasts exposed to TGFβ1 alone. In addition, IL1β decreased the TGFβ1-induced upregulation of lysyl oxidase, an enzyme involved in collagen cross-linking. Furthermore, we found that lung and dermal fibroblasts do not always behave identically towards IL1β. Suppression of COL1A1 by IL1β in the presence of TGFβ1 is more pronounced in lung fibroblasts compared to dermal fibroblasts, whereas a higher upregulation of MMP1 is seen in dermal fibroblasts. The role of IL1β in fibrosis should be reconsidered, and the differences in phenotypical properties of fibroblasts derived from different organs should be taken into account in future anti-fibrotic treatment regimes.