The differentiation of naive CD4(+) T cells into distinct lineages plays critical roles in mediating adaptive immunity or maintaining immune tolerance. In addition to being a first line of defense, the innate immune system also actively instructs adaptive immunity through antigen presentation and immunoregulatory cytokine production. Here we found that sirtuin 1 (SIRT1), a type III histone deacetylase, plays an essential role in mediating proinflammatory signaling in dendritic cells (DCs), consequentially modulating the balance of proinflammatory T helper type 1 (TH1) cells and antiinflammatory Foxp3(+) regulatory T cells (T(reg) cells). Genetic deletion of SIRT1 in DCs restrained the generation of T(reg) cells while driving TH1 development, resulting in an enhanced T-cell-mediated inflammation against microbial responses. Beyond this finding, SIRT1 signaled through a hypoxia-inducible factor-1 alpha (HIF1α)-dependent pathway, orchestrating the reciprocal TH1 and T(reg) lineage commitment through DC-derived IL-12 and TGF-β1. Our studies implicates a DC-based SIRT1-HIF1α metabolic checkpoint in controlling T-cell lineage specification.

Mice have a strong ability to eliminate renal calcium oxalate crystals, and our previous examination indicated a susceptibility in which monocyte-macrophage interaction could participate in the phenomenon. To clarify the macrophage-related factors playing roles in the prevention of crystal formation in mouse kidneys, morphologic and expression studies based on microarray pathway analysis were performed. Eight-week-old male C57BL/6N mice were administered 80 mg/kg of glyoxylate by daily intraabdominal injection for 15 days, and the kidneys were extracted every 3 days for DNA microarray analysis. Based on the raw data of microarray analysis, pathway analyses of inflammatory response demonstrated macrophage activation through the increased expression of chemokine (C-X-C) ligand 1, fibronectin 1, and major histocompatability (MHC) class II. Association analysis of related gene expression values by quantitative reverse transcription polymerase chain reaction (RT-PCR) indicated the high association of chemokine (C-C) ligand 2, CD44, colony-stimulating factor 1, fibronectin 1, matrix gla protein, secreted phosphoprotein 1, and transforming growth factor β1 (TGF-β1) with the amount of both renal crystals and F4/80, a macrophage marker. Immunohistochemically, interstitial macrophages increased during the experimental course, and CD44 and MHC class II were upregulated around crystal-formation sites. Ultrastructural observation of renal macrophages by transmission electron microscopy indicated interstitial macrophage migration with the phagocytosis of crystals. In conclusion, increased expression of inflammation-related genes of renal tubular cells induced by crystal formation and deposition could induce monocyte-macrophage migration and phagocytosis via the interaction of CD44 with osteopontin and fibronectin. Such crystal-removing ability of macrophages through phagocytosis and digestion might become a new target for the prevention of stone formation.

To develop a naturally derived tendon tissue engineering scaffold with the preservation of the native ultrastructure, tensile strength, and biochemical composition of the tendon extracellular matrix (ECM), decellularized tendon slices (DTSs) were prepared using repetitive freeze/thaw of the intact Achilles tendons, frozen section, and nuclease treatment. The DTSs were characterized in the native ultrastructure, mechanical properties, biochemical composition, and cytocompatibility. Histological examination and DNA quantification analysis confirmed that cells were completely removed from tendon tissue by repetitive freeze/thaw in combination with nuclease treatment 12 h. The intrinsic ultrastructure of tendon tissue was well preserved based on scanning electron microscopy examination. The tensile strength of the DTSs was retained 85.62% of native tendon slice. More than 93% of proteoglycans (fibromodulin, biglycan) and growth factors (TGF-β1, IGF-1, VEGF, and CTGF) inherent in tendon ECM were preserved in the DTSs according to ELISA analysis. Furthermore, the DTSs facilitated attachment and repopulation of NIH-3T3 fibroblasts in vitro. Overall, the DTSs are sheet scaffolds with a combination of elemental mechanical strength and tendon ECM bioactive factors that may have many potential applications in tendon tissue engineering.

Mesenchymalization is a cellular and molecular program in which epithelial cells progressively lose their well-differentiated phenotype and adopt mesenchymal characteristics. Tumor mesenchymalization occurs during the progression of cancer to metastatic disease, and is also associated with resistance to multiple therapeutics, including killing by cytotoxic immune cells. Furthermore, tumor cells can evade immune destruction by upregulating the checkpoint molecule PD-L1, and emerging research has found higher PD-L1 expression in mesenchymalized tumors. Here, the association between TGF-β1-mediated mesenchymalization and PD-L1 was investigated in non-small cell lung cancer cells (NSCLC). TGF-β1 was found to upregulate PD-L1 gene transcription in a Smad2-dependent manner, and a positive association between PD-L1 and phosphorylated Smad2 was found in NSCLC tumors. The potential to target these 2 negative immune regulators with a single agent was investigated using M7824, a novel clinical-stage bifunctional agent that targets both PD-L1 and TGF-β. Treatment of NSCLC cells with M7824 in vitro and in vivo attenuated features of TGF-β1-mediated mesenchymalization, including mesenchymal marker expression, proliferation suppression, and chemoresistance. These findings demonstrate that upregulation of tumor cell PD-L1 is a novel mechanism of TGF-β1-induced immunosuppression in NSCLC, and that treatment with M7824 has the potential to simultaneously block both tumor mesenchymalization and PD-L1-dependent immunosuppression.

SIRT1 (silent information regulator 1), a conserved NAD+-dependent histone deacetylase, is closely related with various biological processes. Moreover, the important role of SIRT1 in alcoholic liver disease, nonalcoholic fatty liver and HCC had been widely reported. Recently, a novel role of SIRT1 was uncovered in organ fibrosis diseases. Here, we investigated the inhibitory effect of SIRT1 in liver fibrogenesis. SIRT1 protein was dramatically decreased in CCl4-treated mice livers. Stimulation of LX-2 cells with TGF-β1 also resulted in a significant suppression of SIRT1 protein. Nevertheless, TGF-β1-induced LX-2 cell activation was inhibited by SIRT1 plasmid, and this was accompanied by up-regulation of cell apoptosis-related proteins. Overexpression of SIRT1 also attenuated TGF-β1-induced expression of myofibroblast markers α-SMA and COL1a. However, the important characteristic of the recovery of liver fibrosis is not only the apoptosis of activated stellate cells but also the reversal of the myofibroblast-like phenotype to a quiescent-like phenotype. Restoration of SIRT1 protein was observed in the in vivo spontaneously liver fibrosis reversion model and in vitro MDI (isobutylmethylxanthine, dexamethasone, and insulin)-induced reversed stellate cells, and forced expression of SIRT1 also promoted the reversal of activated stellate cells. Furthermore, lncRNA MALAT1 (metastasis-associated lung adenocarcinoma transcript 1) was increased in liver fibrosis. RNAi-mediated suppression of MALAT1 resulted in a decrease of myofibroblast markers and restoration of SIRT1 protein. These observations suggested that SIRT1 contributed to apoptosis and reversion of activated LX-2 cells and SIRT1 might be regulated by MALAT1 in liver fibrosis. Therefore, SIRT1 could be considered as a valuable therapeutic target for translational studies of liver fibrosis.

Members of the transforming growth factor beta (TGF-β) superfamily are multifunctional cytokines that regulate several cellular processes, including cell cycle arrest, differentiation, morphogenesis, and apoptosis. TGF-β promotes extracellular matrix production and morphological change. Morphogenetic responses to TGF-β include cell migration and epithelial-mesenchymal transition (EMT), which are critical during embryogenesis, development of fibrotic diseases, and the spreading of advanced carcinomas. The purpose of this study was to clarify how TGF-β regulates the fate of retinal pigment epithelial (RPE) cells. TGF-β1 promoted cell cycle progression and phosphorylation of retinoblastoma protein (Rb) in ARPE-19 cells. TGF-β1 induced survivin expression, which in turn stabilized tubulin and Aurora B. RT-PCR and western blot analysis revealed that survivin expression increased in ARPE-19 cells following TGF-β1 treatment. When survivin was depleted, TGF-β1 induced cell cycle arrest and apoptosis and also reduced Rb phosphorylation. In conclusion, the present study shows that induction of EMT in human RPE cells upregulates survivin, leading to survivin-dependent inhibition of cell cycle arrest and apoptosis. Whether cells undergo EMT or apoptosis in response to TGF-β1 is dependent on their cell cycle state, and TGF-β1 regulates the cell cycle via survivin.

Alzheimer's disease (AD) is characterized by progressive cognitive decline, increasingly attributed to neuronal dysfunction induced by amyloid-β oligomers (AβOs). Although the impact of AβOs on neurons has been extensively studied, only recently have the possible effects of AβOs on astrocytes begun to be investigated. Given the key roles of astrocytes in synapse formation, plasticity, and function, we sought to investigate the impact of AβOs on astrocytes, and to determine whether this impact is related to the deleterious actions of AβOs on synapses. We found that AβOs interact with astrocytes, cause astrocyte activation and trigger abnormal generation of reactive oxygen species, which is accompanied by impairment of astrocyte neuroprotective potential in vitro We further show that both murine and human astrocyte conditioned media (CM) increase synapse density, reduce AβOs binding, and prevent AβO-induced synapse loss in cultured hippocampal neurons. Both a neutralizing anti-transforming growth factor-β1 (TGF-β1) antibody and siRNA-mediated knockdown of TGF-β1, previously identified as an important synaptogenic factor secreted by astrocytes, abrogated the protective action of astrocyte CM against AβO-induced synapse loss. Notably, TGF-β1 prevented hippocampal dendritic spine loss and memory impairment in mice that received an intracerebroventricular infusion of AβOs. Results suggest that astrocyte-derived TGF-β1 is part of an endogenous mechanism that protects synapses against AβOs. By demonstrating that AβOs decrease astrocyte ability to protect synapses, our results unravel a new mechanism underlying the synaptotoxic action of AβOs in AD.SIGNIFICANCE STATEMENT Alzheimer's disease is characterized by progressive cognitive decline, mainly attributed to synaptotoxicity of the amyloid-β oligomers (AβOs). Here, we investigated the impact of AβOs in astrocytes, a less known subject. We show that astrocytes prevent synapse loss induced by AβOs, via production of transforming growth factor-β1 (TGF-β1). We found that AβOs trigger morphological and functional alterations in astrocytes, and impair their neuroprotective potential. Notably, TGF-β1 reduced hippocampal dendritic spine loss and memory impairment in mice that received intracerebroventricular infusions of AβOs. Our results describe a new mechanism underlying the toxicity of AβOs and indicate novel therapeutic targets for Alzheimer's disease, mainly focused on TGF-β1 and astrocytes.

A long non-coding RNA (lncRNA), named myocardial infarction associated transcript (MIAT), has been documented to confer risk of myocardial infarction (MI). The aim of this study is to elucidate the pathophysiological role of MIAT in regulation of cardiac fibrosis. In a mouse model of MI, we found that MIAT was remarkably up-regulated, which was accompanied by cardiac interstitial fibrosis. MIAT up-regulation in MI was accompanied by deregulation of some fibrosis-related regulators: down-regulation of miR-24 and up-regulation of Furin and TGF-β1. Most notably, knockdown of endogenous MIAT by its siRNA reduced cardiac fibrosis and improved cardiac function and restored the deregulated expression of the fibrosis-related regulators. In cardiac fibroblasts treated with serum or angiotensin II, similar up-regulation of MIAT and down-regulation of miR-24 were consistently observed. These changes promoted fibroblasts proliferation and collagen accumulation, whereas knockdown of MIAT by siRNA or overexpression of miR-24 with its mimic abrogated the fibrogenesis. Our study therefore has identified MIAT as the first pro-fibrotic lncRNA in heart and unraveled the role of MIAT in the pathogenesis of MI. These findings also promise that normalization of MIAT level may prove to be a therapeutic option for the treatment of MI-induced cardiac fibrosis and the associated cardiac dysfunction.

BACKGROUND

SMAD4 is a gastrointestinal malignancy-specific tumor suppressor gene found mutated in one third of colorectal cancer specimens and half of pancreatic tumors. SMAD4 inactivation by allelic deletion or intragenic mutation mainly occurs in the late stage of human pancreatic ductal adenocarcinoma (PDAC). Various studies have proposed potential SMAD4-mediated anti-tumor effects in human malignancy; however, the relevance of SMAD4 in the PDAC molecular phenotype has not yet been fully characterized.

METHODS

The AsPC-1, CFPAC-1 and PANC-1 human PDAC cell lines were used. The restoration or knockdown of SMAD4 expression in PDAC cells were confirmed by western blotting, luciferase reporter and immunofluorescence assays. In vitro cell proliferation, xenograft, wound healing, quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR), Western blotting, and immunohistochemistry analysis were conducted using PDAC cells in which SMAD4 was either overexpressed or knocked down.

RESULTS

Here, we report that re-expression of SMAD4 in SMAD4-null PDAC cells does not affect tumor cell growth in vitro or in vivo, but significantly enhances cells migration in vitro. SMAD4 restoration transcriptionally activates the TGF-β1/Nestin pathway and induces expression of several transcriptional factors. In contrast, SMAD4 loss in PDAC leads to increased expression of E-cadherin, vascular endothelial growth factor (VEGF), epidermal growth factor receptor (EGFR) and CD133. Furthermore, SMAD4 loss causes alterations to multiple kinase pathways (particularly the phosphorylated ERK/p38/Akt pathways), and increases chemoresistance in vitro. Finally, PDAC cells with intact SMAD4 are more sensitive to TGF-β1 inhibitor treatment to reduced cell migration; PDAC cells lacking SMAD4 showed decreased cell motility in response to EGFR inhibitor treatment.

CONCLUSIONS

This study revealed the molecular basis for SMAD4-dependent differences in PDAC with the aim of identifying the subset of patients likely to respond to therapies targeting the TGF-β or EGFR signaling pathways and of identifying potential therapeutic interventions for PDAC patients with SMAD4 defects.

The Forkhead family of transcription factors comprises numerous members and is implicated in various cellular functions, including cell growth, apoptosis, migration, and differentiation. In this study, we identified the Forkhead factor FoxQ1 as increased in expression during TGF-β1 induced changes in epithelial differentiation, suggesting functional roles of FoxQ1 for epithelial plasticity. The repression of FoxQ1 in mammary epithelial cells led to a change in cell morphology characterized by an increase in cell size, pronounced cell-cell contacts, and an increased expression of several junction proteins (e.g., E-cadherin). In addition, FoxQ1 knock-down cells revealed rearrangements in the actin-cytoskeleton and slowed down cell cycle G1-phase progression. Furthermore, repression of FoxQ1 enhanced the migratory capacity of coherent mammary epithelial cells. Gene expression profiling of NM18 cells indicated that FoxQ1 is a relevant downstream mediator of TGF-β1-induced gene expression changes. This included the differential expression of transcription factors involved in epithelial plasticity, for example, Ets-1, Zeb1, and Zeb2. In summary, this study has elucidated the functional impact of FoxQ1 on epithelial differentiation.

Mesenchymal stem cells (MSCs) have been suggested to participate in immune regulation and airway repair/remodeling. TGF-β1 is critical in the recruitment of stem/progenitor cells for tissue repair, remodeling, and cell differentiation. In this study, we sought to investigate the role of TGF-β1 in MSC migration in allergic asthma. We examined nestin expression (a marker for MSCs) and TGF-β1 signaling activation in airways in cockroach allergen extract (CRE)-induced mouse models. Compared with control mice, there were increased nestin(+) cells in airways and higher levels of active TGF-β1 in serum and p-Smad2/3 expression in lungs of CRE-treated mice. Increased activation of TGF-β1 signaling was also found in CRE-treated MSCs. We then assessed MSC migration induced by conditioned medium from CRE-challenged human epithelium in air/liquid interface culture in Transwell assays. MSC migration was stimulated by epithelial-conditioned medium, but was significantly inhibited by either TGF-β1-neutralizing Ab or TβR1 inhibitor. Intriguingly, increased migration of MSCs from blood and bone marrow to the airway was also observed after systemic injection of GFP(+) MSCs and from bone marrow of Nes-GFP mice following CRE challenge. Furthermore, TGF-β1-neutralizing Ab inhibited the CRE-induced MSC recruitment, but promoted airway inflammation. Finally, we investigated the role of MSCs in modulating CRE-induced T cell response and found that MSCs significantly inhibited CRE-induced inflammatory cytokine secretion (IL-4, IL-13, IL-17, and IFN-γ) by CD4(+) T cells. These results suggest that TGF-β1 may be a key promigratory factor in recruiting MSCs to the airways in mouse models of asthma.

Expression of ADAM12 is low in most normal tissues but is markedly increased in numerous human cancers, including breast carcinomas. We have previously shown that overexpression of ADAM12 accelerates tumor progression in a mouse model of breast cancer (PyMT). In this study, we found that ADAM12 deficiency reduces breast tumor progression in the PyMT model. However, the catalytic activity of ADAM12 seems to be dispensable for its tumor-promoting effect. Interestingly, we show that ADAM12 endogenously expressed in tumor-associated stroma in the PyMT model does not influence tumor progression, but that ADAM12 expression by tumor cells is necessary for tumor progression in these mice. This finding is consistent with our observation that in human breast carcinoma, ADAM12 is almost exclusively located in tumor cells and, only rarely, seen in the tumor-associated stroma. We hypothesized, however, that the tumor-associated stroma may stimulate ADAM12 expression in tumor cells, on the basis of the fact that TGF-β1 stimulates ADAM12 expression and is a well-known growth factor released from tumor-associated stroma. TGF-β1 stimulation of ADAM12-negative Lewis lung tumor cells induced ADAM12 synthesis, and growth of these cells in vivo induced more than 200-fold increase in ADAM12 expression. Our observation that ADAM12 expression is significantly higher in the terminal duct lobular units (TDLU) adjacent to human breast carcinoma compared with TDLUs found in normal breast tissue supports our hypothesis that tumor-associated stroma triggers ADAM12 expression.

The high mortality and disability of diabetic nonhealing skin ulcers create an urgent need for the development of more efficacious strategies targeting diabetic wound healing. In the current study, using human clinical specimens, we show that perilesional skin tissues from patients with diabetes are under more severe oxidative stress and display higher activation of the nuclear factor-E2-related factor 2 (NRF2)-mediated antioxidant response than perilesional skin tissues from normoglycemic patients. In a streptozotocin-induced diabetes mouse model, Nrf2(-/-) mice have delayed wound closure rates compared with Nrf2(+/+) mice, which is, at least partially, due to greater oxidative DNA damage, low transforming growth factor-β1 (TGF-β1) and high matrix metalloproteinase 9 (MMP9) expression, and increased apoptosis. More importantly, pharmacological activation of the NRF2 pathway significantly improves diabetic wound healing. In vitro experiments in human immortalized keratinocyte cells confirm that NRF2 contributes to wound healing by alleviating oxidative stress, increasing proliferation and migration, decreasing apoptosis, and increasing the expression of TGF-β1 and lowering MMP9 under high-glucose conditions. This study indicates an essential role for NRF2 in diabetic wound healing and the therapeutic benefits of activating NRF2 in this disease, laying the foundation for future clinical trials using NRF2 activators in treating diabetic skin ulcers.

Cripto is a small, GPI-anchored signaling protein that regulates cellular survival, proliferation, differentiation and migration during normal developmental processes and tumorigenesis. Cripto functions as an obligatory co-receptor for the TGF-β ligands Nodal, GDF1 and GDF3 but attenuates signaling of others such as activin-A, activin-B and TGF-β1. Soluble, secreted forms of Cripto also activate Src, ras/raf/MAPK and PI3K/Akt pathways via a mechanism that remains largely obscure. This review describes the biological roles and signaling mechanisms of Cripto, highlighting our identification of the 78 kDa glucose regulated protein (GRP78) as a cell surface receptor/co-factor required for Cripto signaling via both TGF-β and Src/MAPK/PI3K pathways. We discuss emerging evidence indicating that Cripto/GRP78 signaling regulates normal somatic stem cells and their tumorigenic counterparts.

The proliferation and epithelial-mesenchymal transition (EMT) of retinal pigment epithelium (RPE) cells are the major pathological changes in development of proliferative vitreoretinopathy (PVR), which leads to severe visual impairment. Histone deacetylases (HDACs)-mediated epigenetic mechanisms play important roles in controlling various physiological and pathological events. However, whether HDACs are involved in the regulation of proliferation and EMT in PRE cells remains unidentified. In this study, we evaluated the expression profile of HDAC family (18 genes) and found that some of class I and class II HDACs were up-regulated in transforming growth factor-β2 (TGF-β2)/TGF-β1-stimulated RPE cells. Tricostatin A (TSA), a class I and II HDAC inhibitor, suppressed the proliferation of RPE cells by G1 phase cell cycle arrest through inhibition of cyclin/CDK/p-Rb and induction of p21 and p27. In the meantime, TSA strongly prevented TGF-β2-induced morphological changes and the up-regulation of α-SMA, collagen type I, collagen type IV, fibronectin, Snail and Slug. We also demonstrated that TSA affected not only the canonical Smad signalling pathway but also the non-canonical TGF-β/Akt, MAPK and ERK1/2 pathways. Finally, we found that the underlying mechanism of TSA affects EMT in RPE cells also through down-regulating the Jagged/Notch signalling pathway. Therefore, this study may provide a new insight into the pathogenesis of PVR, and suggests that epigenetic treatment with HDAC inhibitors may have therapeutic value in the prevention and treatment of PVR.

Peritoneal dissemination including omental metastasis is the most frequent route of metastasis and an important prognostic factor in advanced ovarian cancer. We analyzed the publicly available microarray dataset (GSE2109) using binary regression and found that the transforming growth factor (TGF)-beta signaling pathway was activated in omental metastases as compared to primary sites of disease. Immunohistochemical analysis of TGF-beta receptor type 2 and phosphorylated SMAD2 indicated that both were upregulated in omental metastases as compared to primary disease sites. Treatment of the mouse ovarian cancer cell line HM-1 with recombinant TGF-β1 promoted invasiveness, cell motility and cell attachment while these were suppressed by treatment with A-83-01, an inhibitor of the TGF-β signaling pathway. Microarray analysis of HM-1 cells treated with TGF-β1 and/or A-83-01 revealed that A-83-01 efficiently inhibited transcriptional changes that are induced by TGF-β1. Using gene set enrichment analysis, we found that genes upregulated by TGF-β1 in HM-1 cells were also significantly upregulated in omental metastases compared to primary sites in the human ovarian cancer dataset, GSE2109 (false discovery rate (FDR) q = 0.086). Therapeutic effects of A-83-01 in a mouse model of peritoneal dissemination were examined. Intraperitoneal injection of A-83-01 (150 μg given three times weekly) significantly improved survival (p = 0.015). In summary, these results show that the activated TGF-β signaling pathway in peritoneal metastases is a potential therapeutic target in ovarian cancer.

Three decades ago, the marine-derived compound sinularin was shown to have anti-edematous effects on paw edema induced by carrageenan or adjuvant. To the best of our knowledge, no new studies were conducted to explore the bioactivity of sinularin until we reported the analgesic properties of sinularin based on in vivo experiments. In the present study, we found that sinularin significantly inhibits the upregulation of proinflammatory proteins, inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2) and upregulates the production of transforming growth factor-β (TGF-β) in lipopolysaccharide (LPS)-stimulated murine macrophage RAW 264.7 cells according to western blot analysis. We found that subcutaneous (s.c.) administration of sinularin (80 mg/kg) 1 h before carrageenan injection significantly inhibited carrageenan-induced nociceptive behaviors, including thermal hyperalgesia, mechanical allodynia, cold allodynia, and hindpaw weight-bearing deficits. Further, s.c. sinularin (80 mg/kg) significantly inhibited carrageenan-induced microglial and astrocyte activation as well as upregulation of iNOS in the dorsal horn of the lumbar spinal cord. Moreover, s.c. sinularin (80 mg/kg) inhibited carrageenan-induced tissue inflammatory responses, redness and edema of the paw, and leukocyte infiltration. The results of immunohistochemical studies indicate that s.c. sinularin (80 mg/kg) could upregulate production of TGF-β1 in carrageenan-induced inflamed paw tissue. The present results demonstrate that systemic sinularin exerts analgesic effects at the behavioral and spinal levels, which are associated with both inhibition of leukocyte infiltration and upregulation of TGF-β1.

We present a strategy to conjugate TGF-β1 into fibrin hydrogels to mimic the in vivo presentation of the growth factor in a 3D context. To this end, we engineered fusion proteins between TGF-β1 and a bi-functional peptide composed of a Factor XIII domain and a plasmin cleavage site. In another version the protease cleavage site was omitted to examine whether the growth factor that could not be released from the scaffold by cells had different effects on tissue constructs. The optimal insertion site which yielded correctly processed, functional protein was found between the latency associated peptide and mature TGF-β1 domains. In solution the fusion proteins exhibited similar biological activity as native TGF-β1 as evidenced by inhibition of cell proliferation and promoter activity assays. Immunoprecipitation experiments demonstrated that the fusion TGF-β1 protein bound to fibrinogen in a Factor XIII dependent manner and could be released from the peptide by the action of plasmin. In contrast to bolus delivery, immobilized TGF-β1 induced sustained signaling in fibrin-embedded cells for several days as evidenced by Smad2 phosphorylation. Prolonged pathway activation correlated with enhanced contractile function of vascular constructs prepared from hair follicle mesenchymal stem cells or bone marrow derived smooth muscle cells. Our results suggest that fibrin-immobilized TGF-β1 may be used to enhance the local microenvironment and improve the function of engineered tissues in vitro and potentially also after implantation in vivo where growth factor delivery faces overwhelming challenges.

Aged mice in a murine model of myocardial infarction exhibit less effective myocardial repair. We hypothesized that the deficiency arises from altered lineage choice of endogenous mesenchymal stem cells (MSCs) and faulty maturation of myofibroblasts. Examination of cardiac MSCs revealed a substantial reduction in the pluripotency marker Nanog in cells from aged mice. In addition, the aged MSCs demonstrated a redirected lineage choice that favored adipocytic commitment over fibroblast or myofibroblast differentiation. Fibroblasts derived from aged MSCs demonstrated reduced expression of transforming growth factor-β (TGF-β) receptors I and II and diminished SMAD3 phosphorylation, associated with attenuated contractility and migration. Overexpression of constitutively active TGF-β receptor I in aged cardiac fibroblasts ameliorated their defective motility but did not improve their contractility. Culturing of MSCs and fibroblasts with AICAR (5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside) to activate adenosine monophosphate-activated kinase resulted in TGF-β-dependent development of myofibroblasts with markedly enhanced contractility despite no reduction in adipocytic commitment or increased expression of TGF-β receptors and SMAD3 phosphorylation. The data suggest an adenosine monophosphate-activated kinase-dependent gain of function as mediated by phosphorylation of TGF-β-activated kinase 1 and p38 mitogen-activated protein kinase, which amplifies the response to TGF-β1 via a non-canonical pathway, thus compensating for the reduced expression of TGF-β receptors.

OBJECTIVE

TGF-β is synthesized in an inactive latent complex that is unable to bind to membrane receptors, thus unable to induce a cellular biological response until it has been activated. In addition to activation by chemical mediators, recent studies have demonstrated that mechanical forces may activate latent TGF-βvia integrin-mediated cellular contractions, or mechanical shearing of blood serum. Since TGF-β is present in synovial fluid in latent form, and since normal diarthrodial joint function produces fluid shear, this study tested the hypothesis that the native latent TGF-β1 of synovial fluid can be activated by shearing.

METHODS

Synovial fluid from 26 bovine joints and three adult human joints was sheared at mean shear rates up to 4000 s(-1) for up to 15 h.

RESULTS

Unsheared synovial fluid was found to contain high levels of latent TGF-β1 (4.35 ± 2.02 ng/mL bovine, 1.84 ± 0.89 ng/mL human; mean ± radius of 95% confidence interval) and low amounts (<0.05 ng/mL) of the active peptide. Synovial fluid concentrations of active TGF-β1 increased monotonically with shear rate and shearing duration, reaching levels of 2.64 ± 1.22 ng/mL for bovine and 0.60 ± 0.39 ng/mL for human synovial fluid. Following termination of shearing, there was no statistical change in these active levels over the next 8 h for either species, demonstrating long-term stability of the activated peptide. The unsheared control group continued to exhibit negligible levels of active TGF-β1 at all times.

CONCLUSIONS

Results confirmed the hypothesis of this study and suggest that shearing of synovial fluid might contribute an additional biosynthetic effect of mechanical loading of diarthrodial joints.

OBJECTIVE

We combined two different signal pathways on transforming growth factor β1 (TGF-β1)-Smad and vascular endothelial growth factor C (VEGF-C)/VEGF receptors for exploring changes in pathway members and their influence on lymphangiogenesis and clinicopathological features.

METHODS

Expression of TGF-β1, TGF-βRII, Smad4, VEGF-C, and VEGFR-3 was immunohistochemically evaluated in 147 colon cancer patients who were followed up for 5 years.

RESULTS

Lymphatic vessel density in colon cancer tissues was significantly higher than in normal colonic tissues. Smad4 expression negatively correlated with lymphatic vessel count and VEGF-C expression. VEGF-C expression positively correlated with lymphatic vessel count. Analysis using the Kaplan-Meier method indicated that patients with VEGF-C-positive tumors had significantly shorter overall survival and tumor-free survival time than those with VEGF-C-negative tumors. Patients with Smad4-negative tumors had significantly shorter overall survival and tumor-free survival time than those with Smad4-positive tumors.

CONCLUSIONS

Both Smad4 and VEGF-C are involved in lymphangiogenesis and lymphatic metastasis. Smad4 and VEGF-C expression may be clinically useful indicators for prognostic evaluation in colon cancer patients.

Monocytes emigrate from bone marrow, can infiltrate into brain, differentiate into microglia and clear amyloid β (Aβ) from the brain of mouse models of Alzheimer's disease (AD). Here we show that these mechanisms specifically require CC-chemokine receptor 2 (CCR2) expression in bone marrow cells (BMCs). Disease progression was exacerbated in APP(Swe)/PS1 mice (transgenic mice expressing a chimeric amyloid precursor protein [APPSwe] and human presenilin 1 [PS1]) harboring CCR2-deficient BMCs. Indeed, transplantation of CCR2-deficient BMCs enhanced the mnesic deficit and increased the amount of soluble Aβ and expression of transforming growth factor (TGF)-β1 and TGF-β receptors. By contrast, transplantation of wild-type bone marrow stem cells restored memory capacities and diminished soluble Aβ accumulation in APP(Swe)/PS1 and APP(Swe)/PS1/CCR2⁻/⁻ mice. Finally, gene therapy using a lentivirus-expressing CCR2 transgene in BMCs prevented cognitive decline in this mouse model of AD. Injection of CCR2 lentiviruses restored CCR2 expression and functions in monocytes. The presence of these cells in the brain of non-irradiated APP(Swe)/PS1/CCR2⁻/⁻ mice supports the concept that they can be used as gene vehicles for AD. Decreased CCR2 expression in bone marrow-derived microglia may therefore play a major role in the etiology of this neurodegenerative disease.

Minimal change disease (MCD), the most common idiopathic nephrotic syndrome in children, is characterized by proteinuria and loss of glomerular visceral epithelial cell (podocyte) ultrastructure. Lipopolysaccharide (LPS) and puromycin aminonucleoside (PAN) are used to study podocyte injury in models of MCD in vivo and in vitro. We hypothesized that LPS and PAN influence components of the innate immune system in podocytes such as the Toll-Like Receptor (TLRs), TLR adapter molecules, and associated cytokines. Our results show that cultured human podocytes constitutively express TLRs 1-6 and TLR-10, but not TLRs 7-9. LPS (25 μg/ml) or PAN (60 μg/ml) caused comparable derangement of the actin cytoskeleton in podocytes. Quantitative RT-PCR analysis show that LPS differentially up-regulated the expression of genes for TLRs (1>> 4 ≥ 2>> 3>> 6>> 5), the adapter molecule, MyD88, and transcription factor NF-κB within one hour. LPS also caused increased levels of IL-6, IL-8 and MCP1 without exerting any effect on TNF-α, IFN-α or TGF-β1 at 24 h. Immunofluorescence intensity analysis of confocal microscopy images showed that LPS induced a significant increase in nuclear translocation of NF-κB by 6 h. In contrast, PAN-induced only small changes in the expression of TLRs 2-6 that included a persistent increase in TLRs 2 and 5, a transient increase in TLR-4, and a gradual increase in TLRs 3 and 6 between 1 and 6 h. Correspondingly, it did not alter pro-inflammatory cytokine levels in podocytes. However, PAN induced a low but significant increase in NF-κB nuclear translocation within one hour that remained unchanged up to 6 h. In summary, these novel findings show that LPS, a known TLR-4 ligand, induced the gene expression of multiple TLRs with maximum effect on the expression of TLR-1 suggesting a loss of receptor selectivity and induction of receptor interactions in podocytes. A comparable derangement of the podocyte cytoskeleton and significant increase in the nuclear translocation of NF-κB by PAN suggest that disparate but complementary mechanisms may contribute to the development of podocytopathy in MCD.