BACKGROUND

Mesenchymal stem cells (MSC) are currently strong candidates for cell-based therapies. They are well known for their differentiation potential and immunoregulatory properties and have been proven to be potentially effective in the treatment of a large variety of diseases, including neurodegenerative disorders. Currently there is no treatment that provides consistent long-term benefits for patients with multiple system atrophy (MSA), a fatal late onset α-synucleinopathy. Principally neuroprotective or regenerative strategies, including cell-based therapies, represent a powerful approach for treating MSA. In this study we investigated the efficacy of intravenously applied MSCs in terms of behavioural improvement, neuroprotection and modulation of neuroinflammation in the (PLP)-αsynuclein (αSYN) MSA model.

RESULTS

MSCs were intravenously applied in aged (PLP)-αSYN transgenic mice. Behavioural analyses, defining fine motor coordination and balance capabilities as well as stride length analysis, were performed to measure behavioural outcome. Neuroprotection was assessed by quantifying TH neurons in the substantia nigra pars compacta (SNc). MSC treatment on neuroinflammation was analysed by cytokine measurements (IL-1α, IL-2, IL-4, IL-5, IL-6, IL-10, IL-17, GM-CSF, INFγ, MCP-1, TGF-β1, TNF-α) in brain lysates together with immunohistochemistry for T-cells and microglia. Four weeks post MSC treatment we observed neuroprotection in the SNc, as well as downregulation of cytokines involved in neuroinflammation. However, there was no behavioural improvement after MSC application.

CONCLUSIONS

To our knowledge this is the first experimental approach of MSC treatment in a transgenic MSA mouse model. Our data suggest that intravenously infused MSCs have a potent effect on immunomodulation and neuroprotection. Our data warrant further studies to elucidate the efficacy of systemically administered MSCs in transgenic MSA models.

Breakdown of the inner blood-retinal barrier and the blood-brain barrier is associated with changes in tight and adherens junction-associated proteins that link vascular endothelial cells. This study aimed to test the hypothesis that transforming growth factor (TGF)-β1 increases the paracellular permeability of vascular endothelial monolayers through tyrosine phosphorylation of VE-cadherin and claudin-5. Bovine retinal and human brain capillary endothelial cells were grown as monolayers on coated polycarbonate membranes. Paracellular permeability was studied by measuring the equilibration of (14)C-inulin or fluorescence-labelled dextran. Changes in VE-cadherin and claudin-5 expression were studied by immunocytochemistry (ICC) and quantified by cell-based enzyme linked immunosorbent assays (ELISA). Tyrosine phosphorylation of VE-cadherin and claudin-5 was studied by ICC, immunoprecipitation and Western blotting. We found that exposure of endothelial cells to TGF-β1 caused a dose-dependent increase in paracellular permeability as reflected by increases in the equilibration of (14)C-inulin. This effect was enhanced by the tyrosine phosphatase inhibitor orthovanadate and attenuated by the tyrosine kinase inhibitor lavendustin A. ICC and cell-based ELISA revealed that TGF-β1 induced both dose- and time-dependent decreases in VE-cadherin and claudin-5 expression. Assessment of cell viability indicated that changes in these junction-associated proteins were not due to endothelial death or injury. ICC revealed that tyrosine phosphorylation of endothelial monolayers was greatly enhanced by TGF-β1 treatment, and immunoprecipitation of cell lysates showed increased tyrosine phosphorylation of VE-cadherin and claudin-5. Our results suggest that tyrosine phosphorylation of VE-cadherin and claudin-5 is involved in the increased paracellular permeability of central nervous system-derived vascular endothelium induced by TGF-β1.

The matricellular protein, thrombospondin-1 (TSP-1), is prominently expressed during tissue repair. TSP-1 binds to matrix components, proteases, cytokines, and growth factors and activates intracellular signals through its multiple domains. TSP-1 converts latent transforming growth factor-beta1 (TGF-β1) complexes into their biologically active form. TGF-β plays significant roles in cell-cycle regulation, modulation of differentiation, and induction of apoptosis. Although TGF-β1 is a major inhibitor of proliferation in cultured hepatocytes, the functional requirement of TGF-β1 during liver regeneration remains to be defined in vivo. We generated a TSP-1-deficient mouse model of a partial hepatectomy (PH) and explored TSP-1 induction, progression of liver regeneration, and TGF-β-mediated signaling during the repair process after hepatectomy. We show here that TSP-1-mediated TGF-β1 activation plays an important role in suppressing hepatocyte proliferation. TSP-1 expression was induced in endothelial cells (ECs) as an immediate early gene in response to PH. TSP-1 deficiency resulted in significantly reduced TGF-β/Smad signaling and accelerated hepatocyte proliferation through down-regulation of p21 protein expression. TSP-1 induced in ECs by reactive oxygen species (ROS) modulated TGF-β/Smad signaling and proliferation in hepatocytes in vitro, suggesting that the immediately and transiently produced ROS in the regenerating liver were the responsible factor for TSP-1 induction.

CONCLUSIONS

We have identified TSP-1 as an inhibitory element in regulating liver regeneration by TGF-β1 activation. Our work defines TSP-1 as a novel immediate early gene that could be a potential therapeutic target to accelerate liver regeneration.

TGF-β1 binds receptor II (TβRII) to exert its biological activities but its functional importance in kidney diseases remains largely unclear. In the present study, we hypothesized that TβRII may function to initiate the downstream TGF-β signalling and determine the diverse role of TGF-β1 in kidney injury. The hypothesis was examined in a model of unilateral ureteral obstructive (UUO) nephropathy and in kidney fibroblasts and tubular epithelial cells in which the TβRII was deleted conditionally. We found that disruption of TβRII inhibited severe tubulointerstitial fibrosis in the UUO kidney, which was associated with the impairment of TGF-β/Smad3 signalling, but not with the ERK/p38 MAP kinase pathway. In contrast, deletion of TβRII enhanced NF-κB signalling and renal inflammation including up-regulation of Il-1β and Tnfα in the UUO kidney. Similarly, in vitro disruption of TβRII from kidney fibroblasts or tubular epithelial cells inhibited TGF-β1-induced Smad signalling and fibrosis but impaired the anti-inflammatory effect of TGF-β1 on IL-1β-stimulated NF-κB activation and pro-inflammatory cytokine expression. In conclusion, TβRII plays an important but diverse role in regulating renal fibrosis and inflammation. Impaired TGF-β/Smad3, but not the non-canonical TGF-β signalling pathway, may be a key mechanism by which disruption of TβRII protects against renal fibrosis. In addition, deletion of TβRII also enhances NF-κB signalling along with up-regulation of renal pro-inflammatory cytokines, which may be associated with the impairment of anti-inflammatory properties of TGF-β1.

Matricellular proteins participate in the pathogenesis of chronic kidney diseases. We analyzed glomerular gene expression profiles from patients with proteinuric diseases to identify matricellular proteins contributing to the progression of human nephropathies. Several genes encoding matricellular proteins, such as SPARC, THBS1, and CTGF, were induced in progressive nephropathies, but not in nonprogressive minimal-change disease. Periostin showed the highest induction, and its transcript levels correlated negatively with glomerular filtration rate in both glomerular and tubulointerstitial specimen. In well-preserved renal tissue, periostin localized to the glomerular tuft, the vascular pole, and along Bowman's capsule; no signal was detected in the tubulointerstitial compartment. Biopsies from patients with glomerulopathies and renal dysfunction showed enhanced periostin expression in the mesangium, tubular interstitium, and sites of fibrosis. Periostin staining correlated negatively with renal function. α-smooth muscle actin-positive mesangial and interstitial cells localized close to periostin-positive sites, as indicated by co-immunofluorescence. In vitro stimulation of mesangial cells by external addition of TGF-β1 resulted in robust induction of periostin. Addition of periostin to mesangial cells induced cell proliferation and decreased the number of cells expressing activated caspase-3, a marker of apoptosis. These human data indicate for the first time a role of periostin in glomerular and interstitial injury in acquired nephropathies.

Idiopathic pulmonary fibrosis (IPF) is a devastating disease of unknown cause. Key signaling developmental pathways are aberrantly expressed in IPF. The hedgehog pathway plays a key role during fetal lung development and may be involved in lung fibrogenesis. We determined the expression pattern of several Sonic hedgehog (SHH) pathway members in normal and IPF human lung biopsies and primary fibroblasts. The effect of hedgehog pathway inhibition was assayed by lung fibroblast proliferation and differentiation with and without transforming growth factor (TGF)-β1. We showed that the hedgehog pathway was reactivated in the IPF lung. Importantly, we deciphered the cross talk between the hedgehog and TGF-β pathway in human lung fibroblasts. TGF-β1 modulated the expression of key components of the hedgehog pathway independent of Smoothened, the obligatory signal transducer of the pathway. Smoothened was required for TGF-β1-induced myofibroblastic differentiation of control fibroblasts, but differentiation of IPF fibroblasts was partially resistant to Smoothened inhibition. Furthermore, functional hedgehog pathway machinery from the primary cilium, as well as GLI-dependent transcription in the nucleus, was required for the TGF-β1 effects on normal and IPF fibroblasts during myofibroblastic differentiation. These data identify the GLI transcription factors as potential therapeutic targets in lung fibrosis.

OBJECTIVE

Diminish interleukin-1β (IL-1β) signaling in a model of primary osteoarthritis by RNA interference-based transcript reduction or receptor blockade, and quantify changes incurred on transcript expression of additional mediators.

METHODS

Knees of Hartley guinea pigs were collected at 120 and 180 days of age following injection with viral vectors (N = 4/treatment group/date) at 60 days. Two groups received either adeno-associated viral serotype 5 vector containing a knockdown sequence (TV), or adenoviral vector encoding for IL-1 receptor antagonist protein (Ad-IRAP); treatments were contrasted with opposite knees administered corresponding vector controls. A third group evaluated TV relative to saline-only injected knees. Chondropathy and immunohistochemistry findings were compared to untreated guinea pigs. Transcript expression levels in cartilage were calculated using the comparative CT (2(-ΔΔCT)) method and analyzed by one-way analysis of variance (ANOVA) with pairwise comparisons using Tukey 95% confidence intervals.

RESULTS

Vector transduction was confirmed at both harvest dates. TV and Ad-IRAP, relative to vector controls, significantly decreased IL-1β. Inflammatory mediators [tumor necrosis factor-α (TNF-α), IL-8, interferon-γ (IFN-γ)], and catabolic matrix metalloproteinase 13 (MMP13) were also decreased, while anabolic transforming growth factor-β1 (TGF-β1) was increased. IL-1β was also decreased by TV vs saline, with a decrease in MMP13 and increase TGF-β1; TNF-α, IL-8, and IFN-γ were transiently increased.

CONCLUSIONS

This work confirmed that a reduction in IL-1β signaling was accomplished by either method, resulting in decreased expression of three inflammatory mediators and one catabolic agent, and increased expression of an anabolic molecule. Thus, evidence is provided that IL-1β serves a role in vivo in spontaneous osteoarthritis and that these translational tools may provide beneficial disease modification.

Transforming growth factor-β1 (TGF-β1) induces myofibroblast activation of quiescent aortic valve interstitial cells (AVICs), a differentiation process implicated in calcific aortic valve disease (CAVD). The ubiquity of TGF-β1 signaling makes it difficult to target in a tissue specific manner; however, the serotonin 2B receptor (5-HT(2B)) is highly localized to cardiopulmonary tissues and agonism of this receptor displays pro-fibrotic effects in a TGF-β1-dependent manner. Therefore, we hypothesized that antagonism of 5-HT(2B) opposes TGF-β1-induced pathologic differentiation of AVICs and may offer a druggable target to prevent CAVD. To test this hypothesis, we assessed the interaction of 5-HT(2B) antagonism with canonical and non-canonical TGF-β1 pathways to inhibit TGF-β1-induced activation of isolated porcine AVICs in vitro. Here we show that AVIC activation and subsequent calcific nodule formation is completely mitigated by 5-HT(2B) antagonism. Interestingly, 5-HT(2B) antagonism does not inhibit canonical TGF-β1 signaling as identified by Smad3 phosphorylation and activation of a partial plasminogen activator inhibitor-1 promoter (PAI-1, a transcriptional target of Smad3), but prevents non-canonical p38 MAPK phosphorylation. It was initially suspected that 5-HT(2B) antagonism prevents Src tyrosine kinase phosphorylation; however, we found that this is not the case and time-lapse microscopy indicates that 5-HT(2B) antagonism prevents non-canonical TGF-β1 signaling by physically arresting Src tyrosine kinase. This study demonstrates the necessity of non-canonical TGF-β1 signaling in leading to pathologic AVIC differentiation. Moreover, we believe that the results of this study suggest 5-HT(2B) antagonism as a novel therapeutic approach for CAVD that merits further investigation.

Replacement of the knee meniscus requires a material possessing adequate geometrical and biomechanical properties. Meniscal tissue engineering attempts have been unable to produce tissue with collagen content and biomechanical properties, particularly tensile properties, mimicking native menisci. In an effort to obtain the geometric properties and the maturational growth necessary for the recapitulation of biochemical and, thus, biomechanical properties, a scaffoldless cell-based system, the self-assembly process, was used in conjunction with the catabolic enzyme chondroitinase-ABC and TGF-β1. We show that combinations of these agents resulted in maturational growth as evidenced by synergistic enhancement of the radial tensile modulus by 5-fold and the compressive relaxation modulus by 68%, and additive increases of the compressive instantaneous modulus by 136% and Col/WW by 196%. This study shows that tissue engineering can produce a biomaterial that is on par with the biochemical and biomechanical properties of native menisci.

B cells are generally considered to positively regulate immune responses by producing antigen-specific antibodies. B cells are classified into classical CD5(-) conventional B cells and CD5(+) B1 cells. The latter produce multi-specific autoantibodies and are thought to be involved in autoimmune diseases. However, evidence supporting a B cell negative regulatory function has accumulated over the past 30 years. Multiple reports have suggested that absence, or loss, of regulatory B cells exacerbates symptoms of both allergic (including contact hypersensitivity and anaphylaxis) and autoimmune (such as experimental autoimmune encephalomyelitis, chronic colitis, and collagen-induced arthritis) diseases, and in lupus-like models of autoimmunity. Regulatory B cells are characterized by production of the negative regulatory cytokines, IL-10 and TGF-β. IL-10-producing B cells were the first regulatory B cells to be recognized and were termed 'B1TGF-β-producing regulatory B cell subset, Br3, has been shown to be related to immune tolerance in food allergies. Moreover, forkhead box P3 (Foxp3)-expressing B cells have also been identified in humans and may act as regulatory B cells (Bregs). The functional image of regulatory B cells is similar to that of regulatory T cells. Because of the proliferative and apoptotic responses of Br1 and Br3 cells in immune tolerance in non-IgE-mediated food allergy, reciprocal roles and counter-regulatory mechanisms of Br1 and Br3 responses are also suspected. Additionally, different roles for regulatory B and T cells at different time points during initiation and progression of autoimmune disease are described.

BACKGROUND

Myofibroblasts, a derived subset of fibroblasts especially important in scar formation and wound contraction, have been found at elevated levels in affected Dupuytren's tissues. Transformation of fibroblasts to myofibroblasts is characterized by expression of alpha- smooth muscle actin (α-SMA) and increased production of extracellular matrix (ECM) components, both events of relevance to connective tissue remodeling. We propose that increasing the activation of the cyclic AMP (cAMP)/protein kinase A signaling pathway will inhibit transforming growth factor-beta1 (TGF-β1)-induced ECM synthesis and myofibroblast formation and may provide a means to blunt fibrosis.

METHODS

Fibroblasts derived from areas of Dupuytren's contracture cord (DC), from adjacent and phenotypically normal palmar fascia (PF), and from palmar fascia from patients undergoing carpal tunnel release (CTR; CT) were treated with TGF-β1 (2 ng/ml) and/or forskolin (10 μM) (a known stimulator of cAMP). Total RNA and protein extracted was subjected to real time RT-PCR and Western blot analysis.

RESULTS

The basal mRNA expression levels of fibronectin- extra domain A (FN1-EDA), type I (COL1A2) and type III collagen (COL3A1), and connective tissue growth factor (CTGF) were all significantly increased in DC- and in PF-derived cells compared to CT-derived fibroblasts. The TGF-β1 stimulation of α-SMA, CTGF, COL1A2 and COL3A1 was greatly inhibited by concomitant treatment with forskolin, especially in DC-derived cells. In contrast, TGF-β1 stimulation of FN1-EDA showed similar levels of reduction with the addition of forskolin in all three cell types.

CONCLUSIONS

In sum, increasing cAMP levels show potential to inhibit the formation of myofibroblasts and accumulation of ECM components. Molecular agents that increase cAMP may therefore prove useful in mitigating DC progression or recurrence.

Stem cell function declines with age largely due to the biochemical imbalances in their tissue niches, and this work demonstrates that aging imposes an elevation in transforming growth factor β (TGF-β) signaling in the neurogenic niche of the hippocampus, analogous to the previously demonstrated changes in the myogenic niche of skeletal muscle with age. Exploring the hypothesis that youthful calibration of key signaling pathways may enhance regeneration of multiple old tissues, we found that systemically attenuating TGF-β signaling with a single drug simultaneously enhanced neurogenesis and muscle regeneration in the same old mice, findings further substantiated via genetic perturbations. At the levels of cellular mechanism, our results establish that the age-specific increase in TGF-β1 in the stem cell niches of aged hippocampus involves microglia and that such an increase is pro-inflammatory both in brain and muscle, as assayed by the elevated expression of β2 microglobulin (B2M), a component of MHC class I molecules. These findings suggest that at high levels typical of aged tissues, TGF-β1 promotes inflammation instead of its canonical role in attenuating immune responses. In agreement with this conclusion, inhibition of TGF-β1 signaling normalized B2M to young levels in both studied tissues.

The hormone, relaxin, inhibits aberrant myofibroblast differentiation and collagen deposition by disrupting the TGF-β1/Smad2 axis, via its cognate receptor, Relaxin Family Peptide Receptor 1 (RXFP1), extracellular signal-regulated kinase (ERK)1/2 phosphorylation (pERK) and a neuronal nitric oxide (NO) synthase (nNOS)-NO-cyclic guanosine monophosphate (cGMP)-dependent pathway. However, the signalling pathways involved in its additional ability to increase matrix metalloproteinase (MMP) expression and activity remain unknown. This study investigated the extent to which the NO pathway was involved in human gene-2 (H2) relaxin's ability to positively regulate MMP-1 and its rodent orthologue, MMP-13, MMP-2 and MMP-9 (the main collagen-degrading MMPs) in TGF-β1-stimulated human dermal fibroblasts and primary renal myofibroblasts isolated from injured rats; by gelatin zymography (media) and Western blotting (cell layer). H2 relaxin (10-100 ng/ml) significantly increased MMP-1 (by ~50%), MMP-2 (by ~80%) and MMP-9 (by ~80%) in TGF-β1-stimulated human dermal fibroblasts; and MMP-13 (by ~90%), MMP-2 (by ~130%) and MMP-9 (by ~115%) in rat renal myofibroblasts (all p<0.01 vs untreated cells) over 72 hours. The relaxin-induced up-regulation of these MMPs, however, was significantly blocked by a non-selective NOS inhibitor (L-nitroarginine methyl ester (hydrochloride); L-NAME; 75-100 µM), and specific inhibitors to nNOS (N-propyl-L-arginine; NPLA; 0.2-2 µM), iNOS (1400W; 0.5-1 µM) and guanylyl cyclase (ODQ; 5 µM) (all p<0.05 vs H2 relaxin alone), but not eNOS (L-N-(1-iminoethyl)ornithine dihydrochloride; L-NIO; 0.5-5 µM). However, neither of these inhibitors affected basal MMP expression at the concentrations used. Furthermore, of the NOS isoforms expressed in renal myofibroblasts (nNOS and iNOS), H2 relaxin only stimulated nNOS expression, which in turn, was blocked by the ERK1/2 inhibitor (PD98059; 1 µM). These findings demonstrated that H2 relaxin signals through a RXFP1-pERK-nNOS-NO-cGMP-dependent pathway to mediate its anti-fibrotic actions, and additionally signals through iNOS to up-regulate MMPs; the latter being suppressed by TGF-β1 in myofibroblasts, but released upon H2 relaxin-induced inhibition of the TGF-β1/Smad2 axis.

OBJECTIVE

Pseudoexfoliation (PEX) syndrome/glaucoma is a complex, late-onset disorder of the elastic fiber system. Strong genetic risk is conferred by the lysyl oxidase-like 1 (LOXL1) gene, but additional comodulating factors are necessary for the manifestation of the disease. The aim of this study was to analyze the effect of various PEX-associated pathogenic factors on the genotype-correlated expression of LOXL1 and elastin-related genes.

METHODS

Cultured human Tenon's capsule fibroblasts with high- and low-risk LOXL1 haplotypes were exposed to transforming growth factor (TGF)-β1, interleukin (IL)-6, homocysteine, oxidative stress, hypoxia, or ultraviolet (UV) radiation. Changes in the expression of LOXL1 and elastic constituents of PEX material and TGF-β1 were assessed by quantitative real-time PCR, Western blotting, immunohistochemistry, and electron microscopy.

RESULTS

Treatment of fibroblasts with TGF-β1, oxidative stress, UV light, and hypoxia induced a significant increase in expression levels of LOXL1 and elastic proteins, whereas the effect of IL-6 was limited to induction of elastic constituents. Immunohistochemistry and electron microscopy confirmed an upregulation of LOXL1 and elastic fiber proteins and their assembly into extracellular microfibrillar networks with focal aggregation of microfibrils into PEX-like fibrils on stimulation with TGF-β1 and oxidative stress. Basal and stimulated expression of LOXL1 mRNA and protein was slightly decreased in cells carrying the high-risk compared with the low-risk haplotype of LOXL1, but the differences between groups were statistically not significant.

CONCLUSIONS

The findings support the notion that both genetic and nongenetic fibrogenic factors, particularly TGF-β1 and oxidative stress, may cooperate in the stable accumulation of PEX aggregates.

The mechanisms that mediate the initiation and development of intrahepatic cholangiocarcinoma (ICC) associated with hepatitis B and C virus (HBV and HCV, respectively) infection remain largely unclear. In this study we conditionally coexpressed hepatitis B virus X (HBx) and hepatitis C virus core (HCP) proteins in zebrafish livers, which caused fibrosis and consequently contributed to ICC formation at the age of 3 months. Suppressing the transgene expression by doxycycline (Dox) treatment resulted in the loss of ICC formation. The biomarker networks of zebrafish ICC identified by transcriptome sequencing and analysis were also frequently involved in the development of human neoplasms. The profiles of potential biomarker genes of zebrafish ICC were similar to those of human cholangiocarcinoma. Our data also showed that the pSmad3L oncogenic pathway was activated in HBx and HCP-induced ICC and included phosphorylation of p38 mitogen-activated proteinbase (MAPK) and p44/42 mitogen-activated protein kinase (ERK1/2), indicating the association with transforming growth factor beta 1 (TGF-β1) signaling pathway in ICC. Bile duct proliferation, fibrosis, and ICC were markedly reduced by knockdown of TGF-β1 by in vivo morpholinos injections.

CONCLUSIONS

These results reveal that TGF-β1 plays an important role in HBx- and HCP-induced ICC development. This in vivo model is a potential approach to study the molecular events of fibrosis and ICC occurring in HBV and HCV infection.

Immunomodulatory function of bone marrow derived mesenchymal stem cells in cancer has recently been investigated. But the resident mesenchymal stem cells as whole in cancer and in the breast cancer tissue have not been studied well. In the present work we isolated adipose derived stem cells (ASCs) from breast cancer and normal breast tissues to investigate the expressions of IL-4, IL-10 and transforming growth factor (TGF)-β1 in ASCs and to see if ASCs isolated from patients can modulate the regulatory molecules on peripheral blood lymphocytes. Our results showed that IL-10 and TGF-β1 have significantly higher mRNA expressions in ASCs isolated from breast cancer patients than those from normal individuals (P value <0.05). The culture supernatant of ASCs isolated from breast cancer patients with pathological stage III induced upregulation of the mRNA expression levels of IL-4, TGF-β1, IL-10, CCR4 and CD25 in PBLs. In addition, the percentage of CD4+CD25(high)Foxp3(+) T regulatory cells was increased in vitro. When the same culture supernatant was added to ASCs isolated from normal subjects augmentation of the mRNA expressions of IL-4, IL-10, IL-8, MMP2, VEGF and SDF-1 in normal ASCs was also observed. These data collectively conclude that resident ASCs in breast cancer tissue may have crucial roles in breast tumor growth and progression by inducing regulatory molecules and promoting anti-inflammatory reaction within the tumor microenvironment. Further investigation is required to see if the immune suppression induced by ASCs is an independent property from tumor cells or ASCs gain their immunosuppressive potential from malignant cells.

BACKGROUND

Despite recent progress, therapy for metastatic clear cell renal cell carcinoma (CCRCC) is still inadequate. Dysregulated Notch signaling in CCRCC contributes to tumor growth, but the full spectrum of downstream processes regulated by Notch in this tumor form is unknown.

RESULTS

We show that inhibition of endogenous Notch signaling modulates TGF-β dependent gene regulation in CCRCC cells. Analysis of gene expression data representing 176 CCRCCs showed that elevated TGF-β pathway activity correlated significantly with shortened disease specific survival (log-rank test, p = 0.006) and patients with metastatic disease showed a significantly elevated TGF-β signaling activity (two-sided Student's t-test, p = 0.044). Inhibition of Notch signaling led to attenuation of both basal and TGF-β1 induced TGF-β signaling in CCRCC cells, including an extensive set of genes known to be involved in migration and invasion. Functional analyses revealed that Notch inhibition decreased the migratory and invasive capacity of CCRCC cells.

CONCLUSIONS

An extensive cross-talk between the Notch and TGF-β signaling cascades is present in CCRCC and the functional properties of these two pathways are associated with the aggressiveness of this disease.

BACKGROUND

Pirfenidone is a novel anti-fibrotic and anti-inflammatory agent that inhibits the progression of fibrosis in animal models and in patients with idiopathic pulmonary fibrosis (IPF). We previously showed that pirfenidone inhibits the over-expression of collagen type I and of heat shock protein (HSP) 47, a collagen-specific molecular chaperone, in human lung fibroblasts stimulated with transforming growth factor (TGF)-β1 in vitro. The increased numbers of HSP47-positive type II pneumocytes as well as fibroblasts were also diminished by pirfenidone in an animal model of pulmonary fibrosis induced by bleomycin. The present study evaluates the effects of pirfenidone on collagen type I and HSP47 expression in the human alveolar epithelial cell line, A549 cells in vitro.

METHODS

The expression of collagen type I, HSP47 and E-cadherin mRNAs in A549 cells stimulated with TGF-β1 was evaluated by Northern blotting or real-time PCR. The expression of collagen type I, HSP47 and fibronectin proteins was assessed by immunocytochemical staining.

RESULTS

TGF-β1 stimulated collagen type I and HSP47 mRNA and protein expression in A549 cells, and pirfenidone significantly inhibited this process. Pirfenidone also inhibited over-expression of the fibroblast phenotypic marker fibronectin in A549 cells induced by TGF-β1.

CONCLUSIONS

We concluded that the anti-fibrotic effects of pirfenidone might be mediated not only through the direct inhibition of collagen type I expression but also through the inhibition of HSP47 expression in alveolar epithelial cells, which results in reduced collagen synthesis in lung fibrosis. Furthermore, pirfenidone might partially inhibit the epithelial-mesenchymal transition.

Bone morphogenetic proteins (BMPs) possess osteoinductive activities and are useful for clinical treatments, including bone regeneration. We found that transforming growth factor (TGF)-β1 strongly enhances the osteoinductive activity of BMP-2. Collagen sponges containing 5 μg of BMP-2 were implanted into mouse muscle tissues, after which lump-like masses appeared and grew until day 7. Subsequently, calcification occurred in the lump-like masses by day 14. Addition of 50 ng of TGF-β1 to the BMP-2-containing sponges markedly accelerated the growth of the lump-like masses and resulted in a fivefold increase in total bone volume as compared with BMP-2 alone. The number of osteoblasts in ectopic bone tissues at 14 days after implantation induced by BMP-2+TGF-β1 was twofold greater than that with BMP-2 alone, whereas the number of osteoclasts was decreased by half. On the other hand, TGF-β1 accelerated the differentiation of both osteoblasts and osteoclasts in the early stage (2-7 days after implantation) of ectopic bone formation. We also implanted collagen sponges into bone defects surgically created in mouse calvaria. Sponges containing 2.5 μg of BMP-2 and 25 ng of TGF-β1 caused complete filling of the defects with orthotopic bone, whereas those containing 2.5 μg of BMP-2 alone caused only partial filling. These results suggest that TGF-β1 enhances BMP-2-induced ectopic bone formation by accelerating the growth of lump-like masses, and regulates osteoblast and osteoclast generation. Our findings may contribute to the development of a new treatment method for skeletal disorders.

Tissue engineering utilizes scaffolds containing chondrogenic cells to promote cartilage development at a clinically relevant scale, yet there remains a limited understanding of the optimal conditions for inducing differentiation and matrix production. We investigated how cell density and temporal exposure to chondrogenic factors impacted chondrogenesis of human mesenchymal stem cells (hMSCs) encapsulated in poly(ethylene glycol) diacrylate hydrogels. We found maximal proteoglycan and collagen production in constructs seeded between 10 and 25 × 10(6) cells/mL. Matrix deposition was significantly less per cell in constructs seeded at either higher or lower densities, indicating that paracrine communications may remain important despite loss of direct cell-cell contact. In vitro chondrogenesis of hMSCs was first accomplished using pellet cultures and a defined medium containing transforming growth factor (TGF)-β1 and dexamethasone. The differentiation of hMSCs in hydrogels also required initial exposure to TGF-β1, with no chondrogenic matrix produced in its absence. If TGF-β1 was initially included for at least 7 days, its removal impacted collagen production per cell but also lead to an increase in cell number, such that total collagen deposition was equivalent to controls when TGF-β1 was included for at least 3 weeks. Further, proteoglycan content per construct was higher at 6 weeks after removal of TGF-β1 at any time. In contrast to TGF-β1, dexamethasone was not required for chondrogenesis of hMSCs in hydrogels: there was no difference in matrix deposition between hydrogels cultured with or without dexamethasone. Further, without dexamethasone, SOX9 gene expression was higher during early chondrogenesis and there was a significant reduction in collagen I deposition, suggesting that a more hyaline cartilage phenotype is achieved without dexamethasone. Collagen content at 6 weeks was lower if dexamethasone was excluded after the first 7 days, but was equivalent to control if dexamethasone was included for 2 weeks or longer. Proteoglycan deposition was unaffected by dexamethasone exclusion. These results indicate that modulating exposure to TGF-β1 is beneficial for cell survival/proliferation and matrix production from hMSCs in hydrogels, and that not only is dexamethasone dispensable but also its exclusion may be advantageous for forming hyaline cartilage.

SNAIL, a potent repressor of E-cadherin expression, plays a key role in inducing epithelial-to-mesenchymal transition (EMT) in epithelial cells. During EMT, epithelial cells lose cell polarity and adhesion, and undergo drastic morphological changes acquiring highly migratory abilities. Although there is increasing evidence that EMT is involved in the progression of some human cancers, its significance in the progression of pancreatic cancer remains elusive. In Panc-1, a well-known human pancreatic cancer cell line in which EMT is triggered by TGF-β1 treatment, SNAIL and vimentin are highly expressed, whereas E-cadherin expression is scant. In contrast, another human pancreatic cancer cell line, BxPC3, in which SNAIL expression is not detected, has high levels of E-cadherin expression and does not undergo EMT upon TGF-β1 treatment. After transfecting the SNAIL gene into BxPC3, however, the cells undergo EMT with remarkable alterations in cell morphology and molecular expression patterns without the addition of any growth factors. Furthermore, in an orthotopic transplantation model using SCID mice, SNAIL-transfected BxPC3 displayed highly metastatic and invasive activities. In the immunohistochemical analysis of the tumor derived from the SNAIL-expressing BxPC3, alterations suggestive of EMT were observed in the invasive tumor front. SNAIL enabled BxPC3 to undergo EMT, endowing it with a highly malignant potential in vivo. These results indicate that SNAIL-mediated EMT may be relevant in the progression of pancreatic cancer, and SNAIL could be a molecular target for a pancreatic cancer intervention.

Cardiomyopathy contributes to morbidity and mortality in Duchenne muscular dystrophy (DMD), a progressive muscle-wasting disorder. A major feature of the hearts of DMD patients and the mdx mouse model of the disease is cardiac fibrosis. Connective tissue growth factor (CTGF) is involved in the fibrotic process in many organs. This study utilized the mdx mouse model to assess the role of CTGF and other extracellular matrix components during the development of fibrosis in the dystrophic heart. Left ventricular function of mdx and control mice at 6, 29 and 43 weeks was measured by echocardiography. Young (6 weeks old) mdx hearts had normal function and histology. At 29 weeks of age, mdx mice developed cardiac fibrosis and increased collagen expression. The onset of fibrosis was associated with increased CTGF transcript and protein expression. Increased intensity of CTGF immunostaining was localized to fibrotic areas in mdx hearts. The upregulation of CTGF was also concurrent with increased expression of tissue inhibitor of matrix metalloproteinases (TIMP-1). These changes persisted in 43 week old mdx hearts and were combined with impaired cardiac function and increased gene expression of transforming growth factor (TGF)-β1 and matrix metalloproteinases (MMP-2, MMP-9). In summary, an association was observed between cardiac fibrosis and increased CTGF expression in the mdx mouse heart. CTGF may be a key mediator of early and persistent fibrosis in dystrophic cardiomyopathy.

BACKGROUND

A recent study reported that long non-coding RNA activated by TGF-β (lncRNA-ATB) induced epithelial-mesenchymal transition (EMT) through the transforming growth factor-β (TGF-β)/miR-200s/ZEB axis in hepatocellular carcinoma. Herein, we focused on the clinical significance of lncRNA-ATB in gastric cancer (GC) patients.

METHODS

Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) was performed to examine expression of lncRNA-ATB, miR-200b, and miR-200c in GC tissues (n = 183). Patients were divided into high and low lncRNA-ATB expression groups using a cutoff of lncRNA-ATB/GAPDH ≥0.60 or <0.60 to determine the clinicopathological significance of lncRNA-ATB in GC. Moreover, we evaluated the expression of TGF-β, lncRNA-ATB, miR-200s, and ZEB1 in GC cell lines by qRT-PCR. GC cell lines were treated by recombinant TGF-β1 or TGF-β receptor inhibitor to examine morphologic changes and genetic alterations, such as lncRNA-ATB, miR-200s, and ZEB1 levels, with respect to the EMT phenotype.

RESULTS

The high lncRNA-ATB group experienced a lower overall survival rate compared with the low lncRNA-ATB group, and multivariate analysis indicated that lncRNA-ATB was an independent prognostic factor (hazard ratio 3.50; 95 % CI 1.73-7.44; p = 0.0004). miR-200c levels were lower and ZEB1 levels were higher in the high lncRNA-ATB group than in the low lncRNA-ATB group. Treatment with TGF-β in GC cell lines resulted in morphological EMT changes, upregulation of lncRNA-ATB and ZEB1, and downregulation of miR-200c and CDH1. SB431542 reduced lncRNA-ATB expression.

CONCLUSIONS

LncRNA-ATB plays an important role in EMT to promote invasion and metastasis through the TGF-β/miR-200s/ZEB axis, resulting in a poor prognosis in GC. LncRNA-ATB is a novel biomarker of lncRNA, indicative of a poor prognosis in GC patients.

Intracerebral hemorrhage (ICH) is a devastating form of stroke that results from the rupture of a blood vessel in the brain, leading to a mass of blood within the brain parenchyma. The injury causes a rapid inflammatory reaction that includes activation of the tissue-resident microglia and recruitment of blood-derived macrophages and other leukocytes. In this work, we investigated the specific responses of microglia following ICH with the aim of identifying pathways that may aid in recovery after brain injury. We used longitudinal transcriptional profiling of microglia in a murine model to determine the phenotype of microglia during the acute and resolution phases of ICH in vivo and found increases in TGF-β1 pathway activation during the resolution phase. We then confirmed that TGF-β1 treatment modulated inflammatory profiles of microglia in vitro. Moreover, TGF-β1 treatment following ICH decreased microglial Il6 gene expression in vivo and improved functional outcomes in the murine model. Finally, we observed that patients with early increases in plasma TGF-β1 concentrations had better outcomes 90 days after ICH, confirming the role of TGF-β1 in functional recovery from ICH. Taken together, our data show that TGF-β1 modulates microglia-mediated neuroinflammation after ICH and promotes functional recovery, suggesting that TGF-β1 may be a therapeutic target for acute brain injury.