BACKGROUND: Cardiac fibrosis is the hallmark of all forms of chronic heart disease. Activation and proliferation of cardiac fibroblasts are the prime mediators of cardiac fibrosis. Existing studies show that ROS and inflammatory cytokines produced during fibrosis not only signal proliferative stimuli but also contribute to DNA damage. Therefore, as a prerequisite to maintain sustained proliferation in fibroblasts, activation of distinct DNA repair mechanism is essential.
RESULTS: In this study, we report that TET3, a DNA demethylating enzyme, which has been shown to be reduced in cardiac fibrosis and to exert antifibrotic effects does so not only through its demethylating activity but also through maintaining genomic integrity by facilitating error-free homologous recombination (HR) repair of DNA damage. Using both in vitro and in vivo models of cardiac fibrosis as well as data from human heart tissue, we demonstrate that the loss of TET3 in cardiac fibroblasts leads to spontaneous DNA damage and in the presence of TGF-β to a shift from HR to the fast but more error-prone non-homologous end joining repair pathway. This shift contributes to increased fibroblast proliferation in a fibrotic environment. In vitro experiments showed TET3\'s recruitment to H2O2-induced DNA double-strand breaks (DSBs) in mouse cardiac fibroblasts, promoting HR repair. Overexpressing TET3 counteracted TGF-β-induced fibroblast proliferation and restored HR repair efficiency. Extending these findings to human cardiac fibrosis, we confirmed TET3 expression loss in fibrotic hearts and identified a negative correlation between TET3 levels, fibrosis markers, and DNA repair pathway alteration.
CONCLUSIONS: Collectively, our findings demonstrate TET3\'s pivotal role in modulating DDR and fibroblast proliferation in cardiac fibrosis and further highlight TET3 as a potential therapeutic target.

The multifaceted functions ranging from cellular and developmental mechanisms to inflammation and immunity have rendered TGF-ß signaling pathways as critical regulators of conserved biological processes. Recent studies have indicated that this evolutionary conserved signaling pathway among metazoans contributes to the Drosophila melanogaster anti-nematode immune response. However, functional characterization of the interaction between TGF-ß signaling activity and the mechanisms activated by the D. melanogaster immune response against parasitic nematode infection remains unexplored. Also, it is essential to evaluate the precise effect of entomopathogenic nematode parasites on the host immune system by separating them from their mutualistic bacteria. Here, we investigated the participation of the TGF-ß signaling branches, activin and bone morphogenetic protein (BMP), to host immune function against axenic or symbiotic Heterorhabditis bacteriophora nematodes (parasites lacking or containing their mutualistic bacteria, respectively). Using D. melanogaster larvae carrying mutations in the genes coding for the TGF-ß extracellular ligands Daw and Dpp, we analyzed the changes in survival ability, cellular immune response, and phenoloxidase (PO) activity during nematode infection. We show that infection with axenic H. bacteriophora decreases the mortality rate of dpp mutants, but not daw mutants. Following axenic or symbiotic H. bacteriophora infection, both daw and dpp mutants contain only plasmatocytes. We further detect higher levels of Dual oxidase gene expression in dpp mutants upon infection with axenic nematodes and Diptericin and Cecropin gene expression in daw mutants upon infection with symbiotic nematodes compared to controls. Finally, following symbiotic H. bacteriophora infection, daw mutants have higher PO activity relative to controls. Together, our findings reveal that while D. melanogaster Dpp/BMP signaling activity modulates the DUOX/ROS response to axenic H. bacteriophora infection, Daw/activin signaling activity modulates the antimicrobial peptide and melanization responses to axenic H. bacteriophora infection. Results from this study expand our current understanding of the molecular and mechanistic interplay between nematode parasites and the host immune system, and the involvement of TGF-ß signaling branches in this process. Such findings will provide valuable insight on the evolution of the immune role of TGF-ß signaling, which could lead to the development of novel strategies for the effective management of human parasitic nematodes.

Purpose/Aim of the study: Vascular endothelial growth factor (VEGF)-antagonists are given over long time periods in the clinic, but the long-term effects on retinal pigment epithelium (RPE) cells are not fully investigated. This study aims to investigate these effects with two clinical relevant VEGF antagonists, bevacizumab and aflibercept, on the function of primary RPE cells.Materials and Methods: All tests were conducted with primary porcine RPE. Cells were stimulated with bevacizumab or aflibercept (both 250 µg/ml) for 1 day, 7 days or 4 weeks. Cell viability was tested in MTT Assay. Secretion of TGF-ß was tested in ELISA, phagocytosis in a microscopic assay, migration in a scratch assay, and expression of RPE65 in Western blot. Barrier function was tested for bevacizumab in transwell-cultured cells by measuring transepithelial electrical resistance for up to 3 days.Results: Viability was reduced by both antagonists at all time points tested. TGF-ß secretion was not altered by any treatment. Phagocytosis was not significantly reduced by any treatment. Wound healing ability was not significantly altered by any treatment. The expression of RPE65 was reduced by bevacizumab but not aflibercept after 4 weeks. Transepithelial electrical resistance was not altered.Conclusions: Long-term treatment with anti VEGF may affect viability of RPE cells, and treatment with bevacizumab may have effects on RPE function in long-term treatment.

BACKGROUND: Collagen is the most abundant structural protein in the mammalian connective tissue and represents approximately 30% of animal protein. The current study evaluated the potential capacity of collagen extract derived from Nile tilapia skin in improving the cutaneous wound healing in rats and investigated the underlying possible mechanisms. A rat model was used, and the experimental design included a control group (CG) and the tilapia collagen treated group (TCG). Full-thickness wounds were conducted on the back of all the rats under general anesthesia, then the tilapia collagen extract was applied topically on the wound area of TCG. Wound areas of the two experimental groups were measured on days 0, 3, 6, 9, 12, and 15 post-wounding. The stages of the wound granulation tissues were detected by histopathologic examination and the expression of vascular endothelial growth factor (VEGF), and transforming growth factor (TGF-ß1) were investigated using immunohistochemistry. Moreover, relative gene expression analysis of transforming growth factor-beta (TGF-ß1), basic fibroblast growth factor (bFGF), and alpha-smooth muscle actin (α-SMA) were quantified by real-time qPCR.
RESULTS: The histopathological assessment showed noticeable signs of skin healing in TCG compared to CG. Immunohistochemistry results revealed remarkable enhancement in the expression levels of VEGF and TGF-β1 in TCG. Furthermore, TCG exhibited marked upregulation in the VEGF, bFGF, and α-SMA genes expression. These findings suggested that the topical application of Nile tilapia collagen extract can promote the cutaneous wound healing process in rats, which could be attributed to its stimulating effect on recruiting and activating macrophages to produce chemotactic growth factors, fibroblast proliferation, and angiogenesis.
CONCLUSIONS: The collagen extract could, therefore, be a potential biomaterial for cutaneous wound healing therapeutics.

Background: Caries in the dental pulp result in inflammation and damage to the pulp tissue. During inflammation of the pulp, various inflammatory mediators and growth factors are released, including IL-8, IL-10, TLR-2, VEGF and TGF-β through the NF-kB pathway. In the present study, therapy for pulpal caries was performed through pulp capping by giving a combination of propolis and calcium hydroxide (Ca(OH)2). This treatment was expected to stimulate the formation of reparative dentin as an anti-inflammatory material to prevent pulp tissue damage. Methods: 28 Wistar rats were divided into four groups and treated with Ca(OH)2 with or without the addition of propolis for either 7 or 14 days. Immunohistochemical examination was used to determine the expression of IL-8, IL-10, TLR-2, VEGF, TGF-β in the four treatment groups. Results: The group treated with a combination of propolis and Ca(OH)2 for 7 days showed that the expression of IL-10, IL-8, TLR-2, VEGF, TGF-β increased significantly compared to the treatment group treated with only Ca(OH)2. The expression of IL-10, TLR-2, TGF-β, VEGF increased in the treatment group treated with propolis and Ca(OH)2 for 14 days, while the expression of IL-8 in the decreased significantly. Conclusions: Administration of a combination of propolis and Ca(OH)2 has efficacy in the pulp capping treatment process because it has anti-bacterial and immunomodulatory properties. The results show that it is able to stimulate the process of pulp tissue repair through increased expression of IL-10, TGF-β, VEGF, TLR -2 and decreased expression of IL-8.

The overexpression of GATA-3, T-bet and TGF-ß may theoretically induce IL-4/A, IFN-γ and IL-17A expression, respectively. Whether this also applies to fish is not yet known. The plasmid vectors encoding reporter gene (RFP)-tagged T-bet, GATA-3 and TGF-β were used as overexpression tools, transfected into cells or injected intramuscularly to monitor the expression of IFN-γ, IL-4/13A and IL-17A. In addition, the fish were either experimentally challenged with Vibrio anguillarum (VA group) or Piscirickettsia salmonis (PS group). The reporter gene (RFP) inserted upstream of the GATA-3, T-bet and TGF-ß genes, was observed in muscle cell nuclei and in inflammatory cells after intramuscular (i.m.) injection. PS group: following the injection of GATA-3 and T-bet-encoding plasmids, the expression of GATA-3 and T-bet was high at the injection site. The spleen expression of IFN-γ, following the injection of a T-bet-encoding plasmid, was significantly higher on day 2. VA group: The T-bet and GATA-3-overexpressing fish expressed high T-bet and GATA-3 mRNA levels in the muscles and on day 4 post-challenge. The expression of TGF-ß in the muscles of fish injected with TGF-ß-encoding plasmids was significantly higher on days 7 (8 days pre-challenge) and 19 (4 days after challenge). The protective effects of the overexpression of T-bet, GATA-3 and TGF-ß on both bacterial infections were negligible.

The present review article compares NK cell subsets and cytokine patterns determined in the peripheral blood as well as results of functional in-vitro assays using peripheral NK cells of idiopathic recurrent miscarriage (iRM) patients with corresponding results obtained in female healthy controls and female renal transplant recipients with good long-term graft function. Immune mechanisms, inducing transplant rejection in long-term transplant recipients might also be able to induce rejection of semi-allogeneic fetal cells in patients with iRM. Consequently, the immune status of transplant recipients with good stable long-term graft function should be different from the immune status of iRM patients. iRM patients show a strong persistent cytotoxic NK cell response in the periphery. Simultaneously, immunostimulatory Th1 as well as immunosuppressive Th2 type lymphocytes in the blood are strongly activated but plasma levels of immunosuppressive Th2 type cytokines are abnormally low. In-vitro, unstimulated NK cell cultures of iRM patients show a strong spontaneous TGF-ß1 release in the supernatant but lower TGF-ß1 levels after stimulation with tumor cell line K562, suggesting strong consumption of TGF-ß1 by pre-activated NK cells of iRM patients that might contribute to the low systemic Th2 type plasma levels. iRM patients do not show a systemic switch to a Th2 type cytokine pattern and one might hypothesize that low TGF-ß plasma levels indicate low TGF-ß levels in the micromilieu immediately before fetal rejection. Persistent TGF-ß deficiency implies a persistent unfavorable micromilieu for pregnancy resulting in failing tolerance induction due to lack of TGF-ß, a condition that might contribute to iRM.

Peritoneal fibrosis (PF) represents a long-term complication of peritoneal dialysis (PD), affecting peritoneal membrane (PM) integrity and function. Understanding the mechanisms underlying PF development in an uremic environment aiming alternative therapeutic strategies for treating this process is of great interest. The aim of this study was to analyze the effects of tamoxifen (TAM) and recombinant BMP7 (rBMP7) in an experimental model of PF developed in uremic rats.
To mimic the clinical situation of patients on long-term PD, a combo model, characterized by the combination of PF and CKD with severe uremia, was developed in Wistar rats. PF was induced by intraperitoneal (IP) injections of chlorhexidine gluconate (CG), and CKD was induced by an adenine-rich diet. Uremia was confirmed by severe hypertension, increased blood urea nitrogen (BUN> 120 mg/dL) and serum creatinine levels (> 2 mg/dL). Uremic rats with PF were treated with TAM (10 mg/Kg by gavage) or BMP7 (30 μg/Kg, IP). Animals were followed up for 30 days.
CG administration in uremic rats induced a striking increase in PM thickness, neoangiogenesis, demonstrated by increased capillary density, and failure of ultrafiltration capacity. These morphological and functional changes were blocked by TAM or rBMP7 treatment. In parallel, TAM and rBMP7 significantly ameliorated the PM fibrotic response by reducing α-SMA, extracellular matrix proteins and TGF-ß expression. TAM or rBMP7 administration significantly inhibited peritoneal Smad3 expression in uremic rats with PF, prevented Smad3 phosphorylation, and induced a remarkable up-regulation of Smad7, an intracellular inhibitor of TGFβ/Smad signaling, contributing to a negative modulation of profibrotic genes. Both treatments were also effective in reducing local inflammation, possibly by upregulating IκB-α expression in the PM of uremic rats with PF. In vitro experiments using primary peritoneal fibroblasts activated by TGF-ß confirmed the capacity of TAM or rBMP7 in blocking inflammatory mediators, such as IL-1ß expression.
In conclusion, these findings indicate important roles of TGF-ß/Smad signaling in PF aggravated by uremia, providing data regarding potential therapeutic approaches with TAM or rBMP7 to block this process.

Osteogenesis imperfecta (OI) is a heritable bone fragility disorder that is usually caused by mutations affecting collagen type I encoding genes. Recent studies in mouse models of recessive OI, Crtap-/- mice, and dominant OI, +/G610C mice, found that application of a transforming growth factor beta (TGF-β) neutralizing antibody 1D11 rescues the bone phenotype. In the present study, we investigated TGF-β signaling in a mouse model of severe dominant OI with a high incidence of spontaneous fractures, Col1a1Jrt/+ mice, and the effect of TGF-β neutralizing antibody 1D11 on bone phenotype in 8-week-old mice. Col1a1Jrt/+ mice had elevated TGF-β signaling in bone tissue. Treatment of Col1a1Jrt/+ mice with 1D11 was associated with increased bone length but had no significant effect on bone mass or bone mechanical properties, and no significant treatment-associated differences in serum markers of bone formation (alkaline phosphatase activity) or resorption (tartrate-resistant acid phosphatase) were found. Our data thus indicate that the TGF-β neutralizing antibody 1D11 is not effective in a mouse model of dominant OI with a high incidence of spontaneous fractures. © 2018 American Society for Bone and Mineral Research.

Pluripotent stem cells (PSCs) could provide a powerful system to model development of the human esophagus, whose distinct tissue organization compared to rodent esophagus suggests that developmental mechanisms may not be conserved between species. We therefore established an efficient protocol for generating esophageal progenitor cells (EPCs) from human PSCs. We found that inhibition of TGF-ß and BMP signaling is required for sequential specification of EPCs, which can be further purified using cell-surface markers. These EPCs resemble their human fetal counterparts and can recapitulate normal development of esophageal stratified squamous epithelium during in vitro 3D cultures and in vivo. Importantly, combining hPSC differentiation strategies with mouse genetics elucidated a critical role for Notch signaling in the formation of this epithelium. These studies therefore not only provide an efficient approach to generate EPCs, but also offer a model system to study the regulatory mechanisms underlying development of the human esophagus.