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Articular cartilage is an avascular and flexible connective tissue found in joints. It produces a cushioning effect at the joints and provides low friction to protect the ends of the bones from wear and tear/damage. It has poor repair capacity and any injury can result pain and loss of mobility. Transforming growth factor-beta (TGF-β), a cytokine superfamily, regulates cell function, including differentiation and proliferation. Although the function of the TGF-βs in various cell types has been investigated, their function in cartilage repair is as yet not fully understood. The effect of TGF-β3 in biological regulation of primary chondrocyte was investigated in this work. TGF-β3 provided fibroblastic morphology to chondrocytes and therefore overall reduction in cell proliferation was observed. The length of the cells supplemented with TGF-β3 were larger than the cells without TGF-β3 treatment. This was caused by the fibroblast like cells (dedifferentiated chondrocytes) which occupied larger areas compared to cells without TGF-β3 addition. The healing process of the model wound closure assay of chondrocyte multilayer was slowed down by TGF-β3, and this cytokine negatively affected the strength of chondrocyte adhesion to the cell culture surface., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Abnormal macrophage polarization at the maternal-fetal interface may contribute to the development of Preeclampsia (PE). The reason why macrophage polarization changed in PE is still unclear. Decidual mesenchymal stem cells (dMSCs) could regulate macrophage polarization. However, miRNA in dMSCs of PE were maladjusted. Therefore, we speculated that miRNA may affect dMSC-regulated macrophage polarization. In this study, we found that miR-494-overexpressed dMSCs inhibit M2 macrophage polarization and this inhibitory effect is mediated by miR-494-reduced PGE2 secretion. Furthermore, we proved that miR-494 is induced by TGF-β3. In summary, our findings suggest that the high expression of TGF-β3 in PE decidua stimulates miR-494 in dMSCs and attenuates the regulation of MSC switching the macrophage toward M2 type, contributing to an immune imbalance at maternal-fetal interface., (© 2016 Federation of European Biochemical Societies.)

Mesenchymal cell proliferation is one of the processes in shelf outgrowth. Both all-trans retinoic acid (atRA) and transforming growth factor-β3 (TGF-β3) play an important role in mouse embryonic palate mesenchymal (MEPM) cell proliferation. The cellular effects of TGF-β are mediated by Smad-dependent or Smad-independent pathways. In the present study, we demonstrate that atRA promotes TGF-β3 promoter demethylation and protein expression, but can cause depression of mesenchymal cell proliferation, especially at embryonic day 14 (E14). Moreover, the inhibition of MEPM cell proliferation by atRA results in the downregulation of Smad signaling mediated by transforming growth interacting factor (TGIF). We speculate that the effects of atRA on MEPM cell proliferation may be mediated by Smad pathways, which are regulated by TGIF but are not related to TGF-β3 expression. Finally, the cellular effects of TGF-β3 on MEPM cell proliferation may be mediated by Smad-independent pathways., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Objective: We have recently shown that mesenchymal stem cells (MSCs) embedded in a hyaluronic acid (HA) hydrogel and exposed to chondrogenic factors (transforming growth factor-β3 [TGF-β3]) produce a cartilage-like tissue in vitro. The current objective was to determine if these same factors could be combined immediately prior to implantation to induce a superior healing response in vivo relative to the hydrogel alone., Design: Trochlear chondral defects were created in Yucatan mini-pigs (6 months old). Treatment groups included an HA hydrogel alone and hydrogels containing allogeneic MSCs, TGF-β3, or both. Six weeks after surgery, micro-computed tomography was used to quantitatively assess defect fill and subchondral bone remodeling. The quality of cartilage repair was assessed using the ICRS-II histological scoring system and immunohistochemistry for type II collagen., Results: Treatment with TGF-β3 led to a marked increase in positive staining for collagen type II within defects (P < 0.05), while delivery of MSCs did not (P > 0.05). Neither condition had an impact on other histological semiquantitative scores (P > 0.05), and inclusion of MSCs led to significantly less defect fill (P < 0.05). For all measurements, no synergistic interaction was found between TGF-β3 and MSC treatment when they were delivered together (P > 0.05)., Conclusions: At this early healing time point, treatment with TGF-β3 promoted the formation of collagen type II within the defect, while allogeneic MSCs had little benefit. Combination of TGF-β3 and MSCs at the time of surgery did not produce a synergistic effect. An in vitro precultured construct made of these components may be required to enhance in vivo repair in this model system.

The CCN family of proteins plays important roles in development and homeostasis of bone and cartilage. To understand the role of CCN4 in chondrogenesis, human bone marrow stromal cells (hBMSCs) were transduced with CCN4 adenovirus (adCCN4) or siRNA to CCN4 (siCCN4) in the presence or absence of transforming growth factor-β3 (TGF-β3). Overexpression of CCN4 enhanced TGF-β3-induced SMAD2/3 phosphorylation and chondrogenesis of hBMSCs in an in vitro assay using a micromass culture model. On the other hand, knockdown of CCN4 inhibited the TGF-β3-induced SMAD2/3 phosphorylation and synthesis of cartilage matrix in micromass cultures of hBMSCs. Immunoprecipitation-western blot analysis revealed that CCN4 bound to TGF-β3 and regulated the ability of TGF-β3 to bind to hBMSCs. In vivo analysis confirmed there was a significant decrease in the gene expression levels of chondrocyte markers in cartilage samples from Ccn4-knock out (KO) mice, compared to those from wild type (WT) control. In order to investigate the regenerative properties of the articular cartilage in Ccn4-KO mice, articular cartilage defects were surgically performed in the knee joints of young mice, and the results showed that the cartilage was partially repaired in WT mice, but not in Ccn4-KO mice. In conclusion, these results show, for the first time, that CCN4 has a positive influence on chondrogenic differentiation by modulating the effects of TGF-β3., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Several hormones and growth factors, including adipokines, play important roles during muscle development and regeneration. CTRP3, a paralog of adiponectin, is a member of the C1q and tumor necrosis factor-related protein (CTRP) superfamily. CTRP3 is a novel adipokine previously reported to reduce glucose output in hepatocytes and lower glucose levels in mice models. In the present study, we provide the first evidence for a physiological role of the CTRP3 in myogenesis using C2C12 myoblasts. CTRP3 was expressed in developing skeletal muscle tissues, and the expression level of CTRP3 was increased during myogenic differentiation of C2C12 cells. Recombinant CTRP3 (rCTRP3) promoted the proliferation of undifferentiated C2C12 myoblasts and this response required activation of the extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathway. In contrary, rCTRP3 inhibited myogenic differentiation and fusion of C2C12 cells by suppressing the expression of myogenic marker genes (myogenin and myosin heavy chain). CTRP3 mRNA expression was increased in C2C12 myoblasts treated with transforming growth factor-β3 (TGF-β3), suggesting that TGF-β3 is one of the extracellular factors regulating CTRP3 expression during myogenesis. These results indicate a novel physiological role for CTRP3 during skeletal myogenesis.

Prior findings in vitro of a TGF-β3 dependent mechanism induced by low dose-rate irradiation and resulting in increased radioresistance and removal of low dose hyper-radiosensitivity (HRS) was tested in an in vivo model. DBA/2 mice were given whole-body irradiation for 1 h at low dose-rates (LDR) of 0.3 or 0.03 Gy/h. Serum was harvested and added to RPMI (4% mouse serum and 6% bovine serum).This medium was transferred to reporter cells (T-47D breast cancer cells or T98G glioblastoma cells). The response to subsequent challenge irradiation of the reporter cells was measured by the colony assay. While serum from unirradiated control mice had no effect on the radiosensitivity in the reporter cells, serum from mice given 0.3 Gy/h or 0.03 Gy/h for 1 h removed HRS and also increased survival in response to doses up to 5 Gy. The effect lasted for at least 15 months after irradiation. TGF-β3 neutralizer added to the medium containing mouse serum inhibited the effect. Serum from mice given irradiation of 0.3 Gy/h for 1 h and subsequently treated with iNOS inhibitor 1400W did not affect radiosensitivity in reporter cells; neither did serum from the unirradiated progeny of mice given 1h LDR whole-body irradiation.

The phenotype of the CD4(+) T cells that mediate the CNS pathology in multiple sclerosis is still unclear, and yet a vital question for developing therapies. One of the conundrums is the role of TGF-β in the development of encephalitogenic Th17 cells. In the present study, TGF-β1 and TGF-β3 were directly compared in their capacity to promote the differentiation of myelin-specific Th17 cells that could induce experimental autoimmune encephalomyelitis (EAE). Myelin-specific CD4(+) T cell receptor transgenic cells differentiated with antigen in the presence of IL-6+TGF-β1 or IL-6+TGF-β3 generated T cells that produced robust amounts of IL-17, but were incapable of inducing EAE when transferred into mice. Further analysis of these non-encephalitogenic Th17 cells found that they expressed lower amounts of GM-CSF or IL-23R, both molecules necessary for encephalitogenicity. Thus, TGF-β, irrespective of isoform, negatively regulates the differentiation of encephalitogenic Th17 cells., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Background: The multifunctional transforming growth factor beta (TGF-β) is a glycoprotein that exists in three isoforms. TGF-β3 expression increases in fetal wound healing and reduces fibronectin and collagen I and III deposition, and also improves the architecture of the neodermis which is a combination of blood vessels and connective tissue during wound healing. Fibroblasts are key cells in the wound healing process. TGF-β3 plays a critical role in scar-free wound healing and fibroblast actions in the wound healing process. The aim of this study was to express the TGF-β3 gene (tgf-b3) in human foreskin fibroblasts (HFF's)., Methods: We obtained HFF's from a newborn and a primary fibroblast culture was prepared. The cells were transfected with TGF-β3-pCMV6-XL5 plasmid DNA by both lipofection and electroporation. Expression of TGF-β3 was measured by enzyme-linked immunosorbent assay (ELISA)., Results: The highest TGF-β3 expression (8.3-fold greater than control) was obtained by lipofection after 72 hours using 3 µl of transfection reagent. Expression was 1.4-fold greater than control by electroporation., Conclusions: In this study, we successfully increased TGF-β3 expression in primary fibroblast cells. In the future, grafting these transfected fibroblasts onto wounds can help the healing process without scarring.

To regenerate articular cartilage tissue from degeneration and trauma, synovial mesenchymal stem cells (SMSCs) were used in this study as therapeutic progenitor cells to induce therapeutic chondrogenesis. To accomplish this, chondrocytes pre-transduced with adenoviral vectors carrying the transforming growth factor (TGF) β3 gene were selected as transgenic companion cells and co-cultured side-by-side with SMSCs in a 3D environment to provide chondrogenic growth factors in situ. We adopted a mixed co-culture strategy for this purpose. Transgenic delivery of TGF-β3 in chondrocytes was performed via recombinant adenoviral vectors. The mixed co-culture of SMSCs and transgenic chondrocytes was produced in alginate gel constructs. Gene expression in both SMSCs and chondrocytes were characterized. Biochemical assays in vitro and in vivo showed that release of TGF-ß3 from transgenic chondrocytes not only induced SMSC differentiation into chondrocytic cells but also preserved the chondrocytic phenotype of chondrocytes from suspected dedifferentiation. As a result, this mixed co-culture strategy in conjunction with TGF-ß3 gene delivery could be a promising approach in cartilage tissue engineering., (Copyright © 2012 John Wiley & Sons, Ltd.)

In stem cell-based chondrogenesis for articular cartilage regeneration, TGF-β3 is dosed to the stem cells to drive differentiation into chondrocytic cells. Meanwhile, type I collagen, which is endogenously expressed in some stem cells (e.g., synovium-derived mesenchymal stem cells) and upregulated by TGF-β3, poses a threat to chondrogenesis, as type I collagen may alter the components and stiffness of articular cartilage. Therefore, a wiser strategy would be to feed the cells with TGF-β3 while at the same time silencing the expression of type I collagen. In this chapter, methods for construction of adenoviral vectors and lentiviral vectors having both of the above functions are given. Their transduction into synovium-derived mesenchymal stem cells for articular cartilage engineering and following characterizations are also described.

Polymeric multilayered capsules (PMCs) have found great applicability in bioencapsulation, an evolving branch of tissue engineering and regenerative medicine. Here, we describe the production of hierarchical PMCs composed by an external multilayered membrane by layer-by-layer assembly of poly(L-lysine), alginate, and chitosan. The core of the PMCs is liquified and encapsulates human adipose stem cells and surface-functionalized collagen II-TGF-β3 poly(L-lactic acid) microparticles for cartilage tissue engineering.

Epilepsy in menopausal women presents several challenges in the treatment including an increased risk of seizures due to hormone replacement therapy. We investigated the hypothesis if raloxifene, a selective oestrogen receptor modulator, could be employed to prevent behavioural seizures and morphological alterations in a mouse model mimicking epilepsy in postmenopausal women. Female mice were made ovotoxic by treatment with 4-vinylcyclohexene diepoxide (VCD) to mimic a postmenopausal state. They were then subjected to kainic acid (KA)-induced seizures and neurotoxicity, as assessed by microscopic examination of hippocampus, relevant to human temporal lobe epilepsy. VCD administration (for 15days followed by a drug-free period of 30days) induced ovotoxicity in mice as evidenced by reduced number of primary ovarian follicles. This was accompanied by a 62.4% reduction in serum oestradiol levels. The bone mineral density of ovotoxic mice, however, remained unaffected. Raloxifene (8mg/kg) reduced the seizure severity score in both normal and ovotoxic mice and protected against degeneration induced by KA in the CA3, CA1 sub-fields and hilus of the DG. Hippocampal TGF-β3 levels were not affected by any of the treatments. We show the potential protective role of raloxifene in preventing seizures and neuronal damage in a mouse model mimicking epilepsy in postmenopausal women which was found unrelated to hippocampal TGF-β3. Raloxifene might represent a novel therapeutic option for postmenopausal temporal lobe epileptic woman., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Background: Transforming growth factor-β3 (TGF-β3) plays a central role in mediating secondary palate fusion along the facial midline. However, the mechanisms by which TGF-β3 functions during secondary palate fusion are still poorly understood., Results: We found that mouse cytokeratin 6α and 17 mRNAs were expressed exclusively in the palate medial edge epithelium on embryonic day 14.5, and this expression was completely abolished in Tgf-β3 mutant embryos. In contrast, we found that Jagged2 was initially expressed throughout the palate epithelium, but was specifically down-regulated in the medial edge epithelium during palatal fusion. Jagged2 down-regulation was regulated by TGF-β3, since Jagged2 was persistently expressed in palatal medial edge epithelium in Tgf-β3 null mutant embryos. Moreover, addition of DAPT, a specific inhibitor of Notch signaling, partially rescued the fusion defects in Tgf-β3 null mutant palatal shelves., Conclusions: Based on these results, together with the previous study indicating that the loss of Jagged2 function promotes embryonic oral epithelial fusion, we concluded that TGF-β3 mediates palate fusion in part by down-regulating Jagged2 expression in palatal medial edge epithelium. In addition, cytokeratin 6α and 17 are two TGF-β3 downstream target genes in palate medial edge epithelium differentiation., (© 2014 Wiley Periodicals, Inc.)

Mesenchymal-cell proliferation is the main process in shelf outgrowth. Both all-trans-retinoic acid (atRA) and transforming growth factor-β3 (TGF-β3) play an important role in mouse embryonic palate mesenchymal (MEPM) cell proliferation. In the present study, we investigated the crosstalk between RA and TGF-β signaling in MEPM-cell proliferation. We found that atRA inhibited MEPM-cell proliferation by downregulating TGF-β/Smad signaling and that TGF-β3 treatment was able to antagonize RA signaling. Transforming growth-interacting factor (TGIF) is a transcriptional repressor that suppresses both TGF-β- and retinoid-driven gene transcription. Furthermore, we investigated the role of TGIF in the interaction between both TGF-β and RA signaling in MEPM-cell proliferation. The results showed that both atRA and TGF-β3 significantly increased the expression level of TGIF, and TGIF mediated the negative interaction between TGF-β and RA signaling pathways, which depended on TGIF binding to Smad2 or RARβ (RA receptor beta). Moreover, after deletion of TGIF, both the effects of atRA on TGF-β-dependent protein expression and the effects of TGF-β on RA-dependent protein expression were lost. So we conclude that there is a negative functional interplay of RA and TGF-β signaling mediated by TGIF to modulate MEPM-cell proliferation., (© 2014 Wiley Periodicals, Inc.)

Background: Transforming growth factor beta (TGF-β) plays major roles in tumorigenesis by regulating cell growth, epithelial-to-mesenchymal transition (EMT), migration/invasion and metastasis. The epithelial markers E-cadherin, claudin-3 and claudin-4, commonly decreased in human adenocarcinomas are actually up regulated during ovarian carcinogenesis. In human ovarian cancer TGF-β1 may either suppress or promote tumor progression, but whether other TGF-β isoforms (TGF-β2 and TGF-β3) exert similar effects is not known., Methods: In this study we investigated the ability of the TGF-β isoforms (TGF-β1-3) to induce proliferation and migration by BrdU labeling, scratch wound and trans-filter migration assays in the human serous adenocarcinoma cell-line NIH-OVCAR3. Transepithelial resistance was measured and EMT observed by light-microscopy. Expression of adherens-, tight-junction and EMT-related transcription factors was analyzed by qRT-PCR and immunoblotting., Results: All TGF-β isoforms dose-dependently inhibited NIH-OVCAR3 cell growth, stimulated tumor cell migration with similar efficiency. The mesenchymal marker N-cadherin and claudin-1 expression was induced and occludin down regulated. However, migrating cells retained an epithelial shape and E-cadherin expression. The E-cadherin repressor SNAIL mRNA levels remained low independently of TGF-β1-3 treatment while ZEB1 expression was enhanced., Conclusions: TGF-β1, TGF-β2 and TGF-β3 promote migration of NIH-OVCAR3 ovarian cancer cells independently of cell proliferation and without conversion to a complete EMT phenotype. Epithelial ovarian cancer commonly metastasis to the surrounding tissue or inside the peritoneum rather than invading blood vessels to set distance metastasis. Our result raises the question whether ovarian cancer primarily spread via collective migration than via single cell invasion.

Background: The Autologous Chondrocytes Transplantation (ACT) method is being studied for repair of cartilage diseases. As the chondrocytes dedifferentiated during monolayer culture, three-dimensional cultures are suggested to redifferentiate them. The aim of this study was investigation of the effect of TGF-β3 growth factor on chondrocytes in pellet culture system., Materials and Methods: The chondrocytes were isolated from three human articular cartilages by enzymatic digestion. The cells of the second passage were transferred to pellet culture system. We determined the chondrogenic medium with TGF-β3 as the experimental group and without it as the control group. After 2 weeks, the aggrecan production was investigated using histological and immunohistochemical (IHC) methods., Results: The presence of glycosaminoglycans was proved through Toluiden blue staining. Comparison of IHC results using MATLAB software showed that aggrecan in the experimental group was significantly higher than in the control group (P ≤ 0.05)., Conclusion: The presence of TGF-β3 in the chondrogenic medium could lead to the production of more aggrecan in chondrocytes cultivated in pellet culture system.

Transforming growth factor (TGF) family is well known to induce the chondevrepogenic differentiation of mesenchymal stem cells (MSC). However, the precise signal transduction pathways and underlying factors are not well known. Thus the present study aims to evaluate the possible role of C2 domain in the chondevrepogenic differentiation of human mesenchymal stem cells. To this end, 145 C2 domains in the adenovirus were individually transfected to hMSC, and morphological changes were examined. Among 145 C2 domains, C2 domain of protein kinase C eta (PKCη) was selected as a possible chondevrepogenic differentiation factor for hMSC. To confirm this possibility, we treated TGFβ3, a well known chondevrepogenic differentiation factor of hMSC, and examined the increased-expression of glycosaminoglycan (GAG), collagen type II (COL II) as well as PKCη using PT-PCR, immunocytochemistry and Western blot analysis. To further evaluation of C2 domain of PKCη, we examined morphological changes, expressions of GAG and COL II after transfection of PKCη -C2 domain in hMSC. Overexpression of PKCη-C2 domain induced morphological change and increased GAG and COL II expressions. The present results demonstrate that PKCη involves in the TGF-β3-induced chondevrepogenic differentiation of hMSC, and C2 domain of PKCη has important role in this process.

Although mesenchymal stromal cells (MSCs) have demonstrated great therapeutic potential, the heterogeneity of MSCs may be responsible for the incongruent data obtained in MSC-based preclinical studies and clinical trials. Here, four mouse clonal MSC lines, termed MSC1, MSC2, MSC3, and MSC4, were isolated and extensively characterized. MSC4 cells grew most rapidly and formed colonies of the largest size, whereas MSC3 cells exhibited the slowest growth and formed only a few tiny clusters. MSC4 cells could differentiate into adipocytes, osteoblasts, and chondrocytes in vitro, and more importantly, establish hematopoietic microenvironment in vivo; whereas the other lines displayed uni-adipogenic, osteo-chondrogenic, or non-differentiation potential. All lines were positive for Sca-1, CD106, and CD44; MSC4 was also positive for CD90.2. In terms of immunosuppressive capacity, MSC2, MSC3, and MSC4 cells exerted clear inhibitory effects on lymphocyte proliferation, whereas MSC1 did not. Further investigation revealed that the NO and not the PGE2 pathway may play a role in the different immunomodulatory effects of the cell lines. To clarify the molecular basis of this heterogeneity, we employed RNA sequencing to compare the gene expression profiles of the four subtypes, revealing a relationship between gene expression and variability in subtype function. This study provides novel information about the heterogeneity of MSCs and insight into the selection of optimal cell sources for therapeutic applications., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Purpose: We have previously shown that TGF-β3 (T3) stimulates extracellular matrix (ECM) assembly while maintaining antifibrotic characteristics in a model using human corneal fibroblasts (HCFs). This model, however, requires non-physiological levels of serum. In the current study, we tested whether T3 could stimulate human corneal keratocytes (HCKs) in vitro to assemble a functional ECM, while maintaining their characteristics., Methods: Human corneal keratocytes and HCFs were isolated and cultured using 1% or 10% serum, respectively ±T3. The constructs were processed for indirect immunofluorescence (IF), transmission electron microscopy (TEM), and qRT-PCR, analyzing for keratocyte marker, keratocan, and ECM components, collagen (col) types I, III, and V., Results: Quantitative reverse transcriptase PCR data showed that keratocan, col I, and V were all upregulated in HCKs compared with HCFs, whereas col III was expressed at low levels in HCKs. Transforming growth factor beta 3 stimulation further enhanced the level of change. Without T3, HCK constructs were very thin, approximately 5 μm; however, as with HCFs, upon stimulation with T3, HCK constructs increased in thickness by approximately 5-fold. Cell counts and ECM production revealed that HCKs assembled more ECM per unit area compared with HCFs, and IF revealed downregulation of fibrotic markers, col III, and thrombospondin-1, with T3 stimulation. Transmission electron microscopy data revealed aligned ECM with long fibrils for all conditions except HCK Controls. Human corneal keratocytes+T3 also showed denser collagen fibrils with more consistent fibril diameter., Conclusions: Overall, the data suggests that it is possible to stimulate matrix secretion and assembly by HCKs in vitro by using a single growth factor, T3.

Taking into consideration the previous evidence of revealing the relationship of early life adversity, major depressive disorder (MDD), and stress-linked immunological changes, we recruited 22 MDD patients with childhood trauma exposures (CTE), 21 MDD patients without CTE, and 22 healthy controls without CTE, and then utilized a novel cytokine antibody array methodology to detect potential biomarkers underlying MDD in 120 peripheral cytokines and to evaluate the effect of CTE on cytokine changes in MDD patients. Although 13 cytokines were identified with highly significant differences in expressions between MDD patients and normal controls, this relationship was significantly attenuated and no longer significant after consideration of the effect of CTE in MDD patients. Depressed individuals with CTE (TD patients) were more likely to have higher peripheral levels of those cytokines. Severity of depression was associated with plasma levels of certain increased cytokines; meanwhile, the increased cytokines led to a proper separation of TD patients from normal controls during clustering analyses. Our research outcomes add great strength to the relationship between depression and cytokine changes and suggest that childhood trauma may play a vital role in the co-appearance of cytokine changes and depression., (Copyright © 2013 Elsevier Inc. All rights reserved.)