The regeneration of articular cartilage remains an unresolved question despite the current access to a variety of tissue scaffolds activated with growth factors relevant to this application. Further advances might result from combining more than one of these factors; here, we propose a scaffold composition optimized for the dual delivery of BMP-7 and TGF-β3, two proteins with described chondrogenic activity. First, we tested in a mesenchymal stem cell micromass culture with TGF-β3 whether the exposure to microspheres loaded with BMP-7 would improve cartilage formation. Histology and qRT-PCR data confirmed that the sustained release of BMP-7 cooperates with TGF-β3 towards chondrogenic differentiation. Then, we optimized a scaffold prototype for tissue culture and dual encapsulation of BMP-7 and TGF-β3. The scaffolds were prepared from poly(lactic-co-glycolic acid), and BMP-7/TGF-β3 were loaded as nanocomplexes with heparin and Tetronic 1107. The scaffolds showed the sustained release of both proteins over four weeks, with minimal burst effect. We finally cultured human mesenchymal stem cells on these scaffolds, in the absence of exogenous chondrogenic factor supplementation. The cells cultured on the scaffolds loaded with BMP-7 and TGF-β3 showed clear signs of cartilage formation macroscopically and histologically. RT-PCR studies confirmed a clear upregulation of cartilage markers SOX9 and Aggrecan. In summary, scaffolds encapsulating BMP-7 and TGF-β3 can efficiently deliver a cooperative growth factor combination that drives efficient cartilage formation in human mesenchymal stem cell cultures. These results open attractive perspectives towards in vivo translation of this technology in cartilage regeneration experiments.

Mucus secretion accumulation in the airways may act as a contributing factor for the development of airflow limitation in severe fetal asthma patients. Accumulated evidences showed that transforming growth factor beta (TGF-β) plays a regulatory role in airway remodeling including mucus hyper-secretion in asthma. However, the detailed molecular mechanisms of TGF-β3 induced MUC5AC hyper-expression in airway epithelium remains unclear. Here, we demonstrated the pivotal roles of autophagy in regulation of MUC5AC hyper-production induced by TGF-β3 in airway epithelium. Our experimental data showed that inhibiting autophagy pathway in repeated ovalbumin (OVA) exposed mice exhibited decreased airway hyper-response and airway inflammation, diminishing the expression of Muc5ac and TGF-β3. Furthermore, our studies demonstrated that autophagy was induced upon exposure to TGF-β3 and then mediated MUC5AC hyper-expression by activating the activator protein-1 (AP-1) in human bronchial epithelial cells. Finally, Smad2/3 pathway was involved in TGF-β3-induced MUC5AC hyper-expressions by promoting autophagy. These data indicated that autophagy was required for TGF-β3 induced airway mucous hyper-production, and that inhibition of autophagy exerted therapeutic benefits for TGF-β3 induced airway mucus secretion.

<b>Background:</b> Boar seminal plasma is rich in cytokines, which could influence the capability of spermatozoa to tolerate preservation. <b>Objectives:</b> To evaluate the involvement of boar seminal plasma cytokines in the changes experienced by boar spermatozoa during their storage, either in liquid or frozen state. <b>Materials and Methods:</b> In two separated experiments, semen samples from healthy and fertile boars were split in two aliquots, one centrifuged twice (1,500 ×g for 10 min) to harvest seminal plasma, whereas the other was either commercially extended (3 × 10⁷ sperm/mL) and liquid-stored at 17°C during 144 h (<i>n</i> = 28, Experiment 1) or frozen-thawed using a standard 0.5 mL protocol (<i>n</i> = 27, Experiment 2). Sixteen cytokines were quantified using Luminex xMAP®. Sperm attributes (CASA-evaluated total and progressive motility; flow cytometry-evaluated sperm viability, production of intracellular H₂O₂ and <mml:math><mml:msubsup><mml:mrow>O</mml:mrow><mml:mrow>2</mml:mrow><mml:mrow><mml:mo>•</mml:mo><mml:mo>-</mml:mo></mml:mrow></mml:msubsup></mml:math> and levels of lipid peroxidation in viable spermatozoa) were evaluated either at 0, 72, or 144 h of liquid storage (Experiment 1) or before freezing and at 30- and 150-min post-thawing (Experiment 2). <b>Results:</b> Multiple linear regression models, with Bayesian approach for variable selection, revealed that the anti-inflammatory <em>TGF</em>-β2, <em>TGF</em>-<em>β3</em>, IL-1Ra, and IL-4 and the pro-inflammatory IL-8 and IL-18, predicted changes in sperm motility for liquid-stored semen while the anti-inflammatory IFN-γ was included in the models predicting changes in all sperm attributes for cryopreserved semen. <b>Conclusion:</b> Specific boar seminal plasma cytokines would contribute to modulate the structural and metabolic changes shown by spermatozoa during preservation, either in liquid or frozen state.

The field of cartilage tissue engineering has been evolved in the last decade and a myriad of scaffolding biomaterials and bioactive agents have been proposed. Controlled release of growth factors encapsulated in the polymeric nanomaterials has been of interest notably for the repair of damaged articular cartilage. Here, we proposed an on-chip hydrodynamic flow focusing microfluidic approach for synthesis of alginate nanogels loaded with the transforming growth factor beta 3 (TGF-β3) through an ionic gelation method in order to achieve precise release profile of these bioactive agents during chondrogenic differentiation of mesenchymal stem cells (MSCs). Alginate nanogels with adjustable sizes were synthesized by fine-tuning the flow rate ratio (FRR) in the microfluidic device consisting of cross-junction microchannels. The result of present study showed that the proposed approach can be a promising tool to synthesize bioactive -loaded polymeric nanogels for applications in drug delivery and tissue engineering.

Astrocytes participate in the development and resolution of neuroinflammation in numerous ways, including the release of cytokines and growth factors. Among many, astrocytes release transforming growth factors beta (TGF-β) TGF-β1, TGF-β2 and TGF-β3. TGF-β1 is the most studied isoform, while production and release of TGF-β2 and TGF-β3 by astrocytes have been poorly characterized. Here, we report that purified cultures of hippocampal astrocytes produce mainly TGF-β3 followed by TGF-β2 and TGF-β1. Furthermore, astrocytes release principally the active form of TGF-β3 over the other two. Changes in release of TGF-β were sensitive to the calcineurin (CaN) inhibitor FK506. Starvation had no effect on TGF-β1 and TGF-β3 while TGF-β2 mRNA was significantly up-regulated in a CaN-dependent manner. We further investigated production and release of astroglial TGF-β in Alzheimer's disease-related conditions. Oligomeric β-amyloid (Aβ) down-regulated TGF-β1, while up-regulating TGF-β2 and TGF-β3, in a CaN-dependent manner. In cultured hippocampal astrocytes from 3xTg-AD mice, TGF-β2 and TGF-β3, but not TGF-β1, were up-regulated, and this was CaN-independent. In hippocampal tissues from symptomatic 3xTg-AD mice, TGF-β2 was up-regulated with respect to control mice. Finally, treatment with recombinant TGF-βs showed that TGF-β2 and TGF-β3 significantly reduced PSD95 protein in cultured hippocampal neurons, and this effect was paralleled by conditioned media from Aβ-treated astrocytes or from astrocytes from 3xTg-AD mice. Taken together, our data suggest that TGF-β2 and TGF-β3 are produced by astrocytes in a CaN-dependent manner and should be investigated further in the context of astrocyte-mediated neurodegeneration.

OBJECTIVE

In the treatment of tumorous diseases, scarring often forms after resection or irradiation. Scarring of the buccal mucosa causes difficulty in opening the mouth and mastication, decreasing quality of life. Transforming growth factor (TGF) β3 is an isoform of TGFβ1 that is known to accelerate scarring, although it has different effects on wound healing. TGFβ3 administration into wounds has been associated with improvement in the quality of healing skin in vivo. TGFβ3 is also considered to be an important anti-scarring factor in buccal mucosa. The present study aimed to examine whether TGFβ3 is effective for prevention and treatment of buccal mucosa scarring.

METHODS

Prospective study using an animal model.

METHODS

Thirty Sprague-Dawley rats were involved in this study. We injected 0.5 mL of TGFβ3 (0.005 μg/mL, 0.05 μg/mL, 0.5 μg/mL, 5 μg/mL) or saline was injected into the buccal submucosa. Fifteen minutes after the injection, the mucosa was removed down to the masseter muscle or orbicularis oris muscle layer using a 6-mm biopsy punch. Six weeks after the operation, the buccal mucosae were harvested after euthanasia. Morphologic and histologic examinations were performed.

RESULTS

The administration of 0.5 μg/mL TGFβ3 induced rapid re-epithelialization and suppressed scar formation. In the submucosal layer, favorable restoration of hyaluronic acid and elastin was seen in the TGFβ3-treated groups compared to the saline-treated group.

CONCLUSIONS

TGFβ3 is considered to be effective for better restoration of extracellular matrices of injured buccal mucosa, suggesting a preventative effect of buccal mucosa scarring.

This study examined resting levels of catabolic and anabolic osteokines related to Wnt signaling and their responses to a single bout of plyometric exercise in child and adolescent females. Fourteen premenarcheal girls [10.5 (1.8) y old] and 12 postmenarcheal adolescent girls [15.0 (1.0) y old] performed a plyometric exercise trial. One resting and 3 postexercise blood samples (5 min, 1 h, and 24 h postexercise) were analyzed for sclerostin, dickkopf-1 (DKK-1), osteoprotegerin (OPG), receptor activator of nuclear factor kappa-β ligand (RANKL), and transforming growth factors (TGF-β1, TGF-β2, and TGF-β3). Premenarcheal girls had significantly higher resting sclerostin, TGF-β1, TGF-β2, and TGF-β3 than the postmenarcheal girls, with no significant time effect or group-by-time interaction. DKK-1 was higher in premenarcheal compared with postmenarcheal girls. There was an overall significant DKK-1 decrease from baseline to 1 h postexercise, which remained lower than baseline 24 h postexercise in both groups. There was neither a significant group effect nor group-by-time interaction in OPG, RANKL, and their ratio. RANKL decreased 5 min postexercise compared with baseline and remained significantly lower from baseline 24 h following the exercise. No changes were observed in OPG. OPG/RANKL ratio was significantly elevated compared with resting values 1 h postexercise. In young females, high-impact exercise induces an overall osteogenic effect through a transitory suppression of catabolic osteokines up to 24 h following exercise.

In recent years, statin group drugs have been widely investigated in treatment of periodontal diseases due to their anti-inflammatory effect. The efficacy of statins can be enhanced by local administration into the periodontal pocket by appropriate delivery systems. The aim of our study was to develop a bioadhesive delivery system for local delivery of atorvastatin in treatment of periodontal disease. For this purpose, gel formulations were prepared using different types of chitosan (base and water soluble) and viscosity, bioadhesivity and syringeability of the gels as well as in vitro drug release properties were investigated vitro. Furthermore, anti-inflammatory effect of the formulations was studied in vitro using tumor necrosis factor (TNF)-alfa induced human gingival fibroblast (hGF) cells. Release of proinflammatory (IL-1β, IL-6, IL-8) and anti-inflammatory (TGF-β1, TGF-β2, TGF-β3, IL-10) cytokines were measured after incubating the hGF cells with the formulations. The viscosity of the formulations was found to be suitable for a local application into periodontal pocket. In presence of drug, bioadhesive property of the formulations was found to increase, and bioadhesion force was within the range, which would retain the delivery system at the application site, subsequently maintain drug levels at desired amount for longer period of time. The release of atorvastatin from the gels was found to be slower than that of the solution. The cytokine levels were found to decrease following application of the formulations, and anti-inflammatory effect was observed to enhance in presence of chitosan. No significant differences were found between base and water-soluble chitosan.

CCN family member 2 (CCN2) has been shown to promote the proliferation and differentiation of chondrocytes, osteoblasts, osteoclasts, and vascular endothelial cells. In addition, a number of growth factors and cytokines are known to work in harmony to promote the process of chondrogenesis and chondrocyte differentiation toward endochondral ossification. Earlier we showed that CCN2 physically interacts with some of them, suggesting that multiple effects of CCN2 on various differentiation stages of chondrocytes may be attributed to its interaction with these growth factors and cytokines. However, little is known about the functional interaction occurring between CCN2 and other growth factors and cytokines in promoting chondrocyte proliferation and differentiation. In this study we sought to shed light on the binding affinities between CCN2 and other essential growth factors and cytokines known to be regulators of chondrocyte differentiation. Using the surface plasmon resonance assay, we analyzed the dissociation constant between CCN2 and each of the following: TGF-β1, TGF-β3, IGF-I, IGF-II, PDGF-BB, GDF5, PTHrP, and VEGF. We found a strong association between CCN2 and VEGF, as well as a relatively high association with TGF-β1, TGF-β3, PDGF-BB, and GDF-5. However, the sensorgrams obtained for possible interaction between CCN2 and IGF-I, IGF-II or PTHrP showed no response. This study underlines the correlation between CCN2 and certain other growth factors and cytokines and suggests the possible participation of such interaction in the process of chondrogenesis and chondrocyte differentiation toward endochondral ossification.

Transforming growth factor (TGF)-β contributes to the malignant phenotype of glioblastoma by promoting invasiveness and angiogenesis and creating an immunosuppressive microenvironment. So far, TGF-β1 and TGF-β2 isoforms have been considered to act in a similar fashion without isoform-specific function in glioblastoma. A pathogenic role for TGF-β3 in glioblastoma has not been defined yet. Here, we studied the expression and functional role of endogenous and exogenous TGF-β3 in glioblastoma models. TGF-β3 mRNA is expressed in human and murine long-term glioma cell lines as well as in human glioma-initiating cell cultures with expression levels lower than TGF-β1 or TGF-β2 in most cell lines. Inhibition of TGF-β3 mRNA expression by ISTH2020 or ISTH2023, two different isoform-specific phosphorothioate locked nucleic acid (LNA)-modified antisense oligonucleotide gapmers, blocks downstream SMAD2 and SMAD1/5 phosphorylation in human LN-308 cells, without affecting TGF-β1 or TGF-β2 mRNA expression or protein levels. Moreover, inhibition of TGF-β3 expression reduces invasiveness in vitro Interestingly, depletion of TGF-β3 also attenuates signaling evoked by TGF-β1 or TGF-β2 In orthotopic syngeneic (SMA-560) and xenograft (LN-308) in vivo glioma models, expression of TGF-β3 as well as of the downstream target, plasminogen-activator-inhibitor (PAI)-1, was reduced, while TGF-β1 and TGF-β2 levels were unaffected following systemic treatment with TGF-β3 -specific antisense oligonucleotides. We conclude that TGF-β3 might function as a gatekeeper controlling downstream signaling despite high expression of TGF-β1 and TGF-β2 isoforms. Targeting TGF-β3in vivo may represent a promising strategy interfering with aberrant TGF-β signaling in glioblastoma. Mol Cancer Ther; 16(6); 1177-86. ©2017 AACR.

The aim of the present was to evaluate the effects of DNA methylation and histone acetylation on 2,3,7,8‑tetrachlorodibenzo‑p‑dioxin (TCDD)‑induced cleft palate in fetal mice. Pregnant mice (n=10) were randomly divided into two groups: i) TCDD group, mice were treated with 28 µg/kg TCDD on gestation day (GD) 10 by oral gavage; ii) control group, mice were treated with an equal volume of corn oil. On GD 16.5, the fetal mice were evaluated for the presence of a cleft palate. An additional 36 pregnant mice were divided into the control and TCDD groups, and palate samples were collected on GD 13.5, GD 14.5 and GD 15.5, respectively. Transforming growth factor‑β3 (TGF‑β3) mRNA expression, TGF‑β3 promoter methylation, histone acetyltransferase (HAT) activity and histone H3 (H3) acetylation in the palates were evaluated in the two groups. The incidence of a cleft palate in the TCDD group was 93.55%, and no cases of cleft palate were identified in the control group. On GD 13.5 and GD 14.5, TGF‑β3 mRNA expression, HAT activity and acetylated H3 levels were significantly increased in the TCDD group compared with the control. Methylated bands were not observed in the TCDD or control groups. In conclusion, at the critical period of palate fusion (GD 13.5‑14.5), TCDD significantly increased TGF‑β3 gene expression, HAT activity and H3 acetylation. Therefore, histone acetylation may be involved in TCDD‑induced cleft palate formation in fetal mice.

The aim of this study was to investigate the protective effect and underlying mechanisms of Ganoderma atrum polysaccharide (PSG-1) on cyclophosphamide (Cy)-induced intestinal mucosal dysfunction in mice. Results showed that PSG-1 promoted the formation of IgA-secreting cells, modulated sIgA, IgE, IgG, IgM secretion, and improved TLR-2, TLR-4, TLR-6 mRNA levels while these factors were suppressed after Cy treatment. CD4⁺ and CD8⁺ T cell numbers were also elevated by PSG-1. Cytokines including IFN-γ, TNF-α, IL-2, IL-12p70, IL-4, IL-1β, IL-17, IL-21, IL-23, TGF-β3 and transcription factors including T-bet, GATA-3, RORγt, Foxp3 increased after PSG-1 administration. Besides, PSG-1 reversed goblet cell numbers, and upregulated tight junction proteins like ZO-1, occludin and claudin-1 in immunosuppressed mice. Apart from these, the autophagy-related proteins LC3, Beclin-1, Atg5 and Atg7 were enhanced by PSG-1. These findings demonstrated that PSG-1 could ameliorate Cy-induced impairment of intestinal immunity and mucosal integrity, which maybe associated with autophagy in mice.

UNASSIGNED

The transforming growth factor (TGF)-β pathway is a well-described inducer of immunosuppression and can act as an oncogenic factor in advanced tumors. Several preclinical and clinical studies show that the TGF-β pathway can be considered a promising molecular target for cancer therapy. The human genome has three TGF-β isoforms and not much is known about the oncogenic response to each of the isoforms. Here, we studied the antitumor response to ISTH0047, a recently developed locked nucleic acid-modified antisense oligonucleotide targeting TGF-β2.

UNASSIGNED

We have studied the anticancer response to ISTH0047 using gymnotic delivery in tumor cell cultures and in in vivo preclinical orthotopic mouse models for primary tumors (breast and kidney tumors) and lung metastasis.

UNASSIGNED

We observed that ISTH0047 is able to significantly reduce TGF-β2 mRNA and protein levels without altering the levels of TGF-β1 and TGF-β3. ISTH0047 prevented lung metastasis in syngeneic orthotopic renal cell carcinoma (RENCA) and breast cancer (4T1) tumor models. In addition, using an orthotopic xenograft model of a lung cancer cell line (CRL5807) that mainly expresses TGF-β2, we observed that ISTH0047 had an important effect on the lung microenvironment inhibiting the growth of lung lesions. ISTH0047 treatment re-educated macrophages in the lung parenchyma to express the tumor-suppressive factor, CD86.

UNASSIGNED

Overall, our data point to TGF-β2 as a therapeutic target and ISTH0047 as a novel anticancer drug to prevent lung metastasis by impacting on the tumor niche, in part, through the induction of CD86 in tumor-associated macrophages.

Cell-loaded hydrogels are frequently applied in cartilage tissue engineering for their biocompatibility, ease of application, and ability to conform to various defect sites. As a bioactive adjunct to the biomaterial, transforming growth factor beta (TGF-β) has been shown to be essential for cell differentiation into a chondrocyte phenotype and maintenance thereof, but the low amounts of endogenous TGF-β in the in vivo joint microenvironment necessitate a mechanism for controlled delivery and release of this growth factor. In this study, TGF-β3 was directly loaded with human bone marrow-derived mesenchymal stem cells (MSCs) into poly-d,l-lactic acid/polyethylene glycol/poly-d,l-lactic acid (PDLLA-PEG) hydrogel, or PDLLA-PEG with the addition of hyaluronic acid (PDLLA/HA), and cultured in vitro. We hypothesize that the inclusion of HA within PDLLA-PEG would result in a controlled release of the loaded TGF-β3 and lead to a robust cartilage formation without the use of TGF-β3 in the culture medium. ELISA analysis showed that TGF-β3 release was effectively slowed by HA incorporation, and retention of TGF-β3 in the PDLLA/HA scaffold was detected by immunohistochemistry for up to 3 weeks. By means of both in vitro culture and in vivo implantation, we found that sulfated glycosaminoglycan production was higher in PDLLA/HA groups with homogenous distribution throughout the scaffold than PDLLA groups. Finally, with an optimal loading of TGF-β3 at 10 μg/mL, as determined by RT-PCR and glycosaminoglycan production, an almost twofold increase in Young's modulus of the construct was seen over a 4-week period compared to TGF-β3 delivery in the culture medium. Taken together, our results indicate that the direct loading of TGF-β3 and stem cells in PDLLA/HA has the potential to be a one-step point-of-care treatment for cartilage injury. STATEMENT OF SIGNIFICANCE: Stem cell-seeded hydrogels are commonly used in cell-based cartilage tissue engineering, but they generally fail to possess physiologically relevant mechanical properties suitable for loading. Moreover, degradation of the hydrogel in vivo with time further decreases mechanical suitability of the hydrogel due in part to the lack of TGF-β3 signaling. In this study, we demonstrated that incorporation of hyaluronic acid (HA) into a physiologically stiff PDLLA-PEG hydrogel allowed for slow release of one-time preloaded TGF-β3, and when loaded with adult mesenchymal stem cells and cultured in vitro, it resulted in higher chondrogenic gene expression and constructs of significantly higher mechanical strength than constructs cultured in conventional TGF-β3-supplemented medium. Similar effects were also observed in constructs implanted in vivo. Our results indicate that direct loading of TGF-β3 combined with HA in the physiologically stiff PDLLA-PEG hydrogel has the potential to be used for one-step point-of-care treatment of cartilage injury.

Articular cartilage injury can result in chondrocyte loss and diminishment of specialised extracellular matrix, which can progress to an osteoarthritic (OA) phenotype. Stem cells have emerged as a favourable approach for articular cartilage regeneration. Identification of miRNAs which influence stem cell fate offers new approaches for application of miRNAs to regenerate articular cartilage. Skeletal stem cells (SSCs) isolated from human bone marrow were cultured as high density micromass' using TGF-β3 to induce chondrogenesis. qPCR and TaqMan qPCR were used to assess chondrogenic gene and miRNA expression. Target prediction algorithms identified potential targets of miR-146b. Transient transfection with miR-146b mimic and western blotting was used to analyse SOX5. Human OA articular chondrocytes were examined for miR-146b expression. Chondrogenic differentiation of human bone marrow derived SSCs resulted in significant down-regulation of miR-146b. Gain of miR-146b function resulted in down-regulation of SOX5. MiR-146b expression was up-regulated in OA chondrocytes. These findings demonstrate the functional role of miR-146b in the chondrogenic differentiation of human bone marrow derived SSCs. MiR-146b may play a role in the pathophysiology of OA. Application of miR-146b combined with stem cell therapy could enhance regeneration of cartilaginous tissue and serve as a potential therapeutic target in the treatment of OA.

Keratoconus (KC) is a complex corneal dystrophy with multifactorial etiology. Previous studies have shown evidence of mitochondrial abnormalities in KC; however, the exact cause of these abnormalities remains unknown. The aim of this study was to identify if transforming growth factor-β (TGF-β) isoforms play a role in the regulation of mitochondrial proteins in human KC cells (HKC).

Human corneal fibroblasts (HCF) and HKC were isolated and cultured for 4 weeks in three different conditions: (a)

MEM + 10%FBS, (b) MEM + 10%FBS + TGF-β1 and (c) MEM + 10%FBS + TGF-β3. All samples were processed for mitochondrial damage analysis using real-time PCR.

We quantified and analyzed 84 mitochondrial and five housekeeping genes in HCFs and HKCs. Our data showed that when TGF-β1 and/or TGF-β3 were compared with control in HCFs, nine genes were significantly different; however, no genes were significantly regulated by the TGF-β isoforms in HKCs. Significant differences were also seen in seven genes when HFCs were compared with HKCs, in all three conditions.

Overall, our data support the growing consensus that mitochondrial dysfunction is a key player in KC disease. These in vitro data show clear links between mitochondrial function and TGF-β isoforms, with TGF-β1 severely disrupting KC-mitochondrial function, while TGF-β3 maintained it, thus suggesting that TGF-β may play a role in KC-disease treatment.

OBJECTIVE

Recent studies have shown encouraging progress toward the use of autogenic and allogenic mesenchymal stem cells (MSCs) to arrest, or even lead to partial regeneration in, intervertebral disc (IVD) degeneration. However, this technology is still in its infancy, and further development is required. The aim of this study was to analyze whether rat adipose-derived mesenchymal stem cells (ADMSC) can differentiate towards IVD-like cells after treatment with transforming growth factor β3 (TGF-β3) in vitro. We also performed quantitative analysis of gene expression for ADMSC only, ADMSCs treated with TGF-β3, and co-cultured ADMSCs treated with TGF-β3.

METHODS

ADMSCs were sub-cultured to homogeneity and used in fluorocytometry assays for CD11, CD45, and CD90/Thy1. ADMSCs were differentiated in spheroid culture towards the chondrogenic lineage by the presence of TGF-β3, dexamethasone, and ascorbate. We also co-cultured pure ADMSCs and nucleus pulposus cells in 24-well plates, and performed immunohistochemical staining, western blotting, and RT-PCR for quantitativeanalysis of gene expression.

RESULTS

Results of fluorocytometry were positive for CD90/Thy1 and negative for CD11 and CD45. TGF-β3-mediated induction of ADMSCs led to the expression of the differentiation markers of intervertebral disc-like cells, such as aggrecan, collagen II, and sox-9. Co-cultured ADMSCs treated with TGF-β3 showed higher expression of differentiation markers and greater extracellular matrix production compared with ADMSCs treated with TGF-β3 alone.

CONCLUSIONS

ADMSC treated with TGF-β3 may be an attractive source for regeneration therapy in degenerative IVD. These findings may also help elucidate the pathologic mechanism of MSC therapy in the degeneration of IVD in vivo.

BACKGROUND

Pseudoexfoliation syndrome (PEXS) may go along with capsular bag shrinkage and luxation. In the present study, we focus on an association of isoforms of TGF-β with capsular bag luxation.

METHODS

Aqueous humor was collected intraoperatively from 20 healthy controls and from 73 otherwise healthy patients with PEXS [PEXS without complications (PEX, n = 33), late PEXS with glaucoma (PEXG, n = 30) and with IOL and capsular bag luxation (PEXL, n = 10)]. The concentrations of TGF-β1, TGF-β2 and TGF-β3 were compared using the Bio-Plex® multiplex beads system based on the non-parametric Kruskal-Wallis H test (p < 0.01).

RESULTS

Concentrations of TGF-β 1, TGF-β 2 and TGF-β 3 were higher in the sub-groups PEX and PEXG than in controls (TGF-β 1; p = 0.009 and 0.0005; TGF-β 2; p = 0.002 and 0.005 and TGF-β 3; 0.0005 and 0.0005; respectively), whereas for TGF β2, no significant difference between controls and PEXL was revealed (p = 1.0). TGF-β2 concentrations were elevated in a similar degree in early PEX and PEXG, but not in PEXL compared to controls (p = 0.002). The concentrations of of TGF-β 1 and TGF-β 3 increased in parallel with the progression of disease. The levels of TGF-β 3, however, did not attain pathophysiological levels (>100 pg/ml) in any group.

CONCLUSIONS

A stage-dependent increase in the concentrations of TGF-β1 and TGF-β3, but not of TGF-β2, accords to the shrinkage of the capsular bag. This could increase the tension on the zonular fibers and contribute to luxation of the capsular bag.

Transforming growth factor-β (TGF-β) is a multifunctional cytokine with important roles in embryogenesis and maintaining tissue homeostasis during adult life. There are three isoforms of TGF-β, i.e., TGF-β1, -β2, and -β3, which signal by binding to a complex of transmembrane type I and type II serine/threonine kinase receptors and intracellular Smad transcription factors. In most cell types TGF-β signals via TGF-β type II receptor (TβRII) and TβRI, also termed activin receptor-like kinase 5 (ALK5). In endothelial cells, TGF-β signals via ALK5 and ALK1. These two type I receptors mediate opposite cellular response for TGF-β. The co-receptor endoglin, highly expressed on proliferating endothelial cells, facilitates TGF-β/ALK1 and inhibits TGF-β/ALK5 signaling. Knockout of TGF-β receptors in mice all result in embryonic lethality during midgestation from defects in angiogenesis, illustrating the pivotal role of TGF-β in this process. This chapter introduces methods for examining the function and regulation of TGF-β in angiogenesis in in vitro assays using cultured endothelial cells and ex vivo metatarsal explants.

Corneal epithelial regeneration through ex vivo expansion of limbal stem cells (LSCs) on 3T3-J2 fibroblasts has revealed some limitations mainly due to the corneal microenvironment not being properly replicated, thus affecting long term results. Insights into the feeder cells that are used to expand LSCs and the mechanisms underlying the effects of human feeder cells have yet to be fully elucidated. We recently developed a standardized methodology to expand human Tenon's fibroblasts (TFs). Here we aimed to investigate whether TFs can be employed as feeder cells for LSCs, characterizing the phenotype of the co-cultures and assessing what human soluble factors are secreted. The hypothesis that TFs could be employed as alternative human feeder layer has not been explored yet. LSCs were isolated from superior limbus biopsies, co-cultured on TFs, 3T3-J2 or dermal fibroblasts (DFs), then analyzed by immunofluorescence (p63α), colony-forming efficiency (CFE) assay and qPCR for a panel of putative stem cell and epithelial corneal differentiation markers (KRT3). Co-cultures supernatants were screened for a set of soluble factors. Results showed that the percentage of p63α(+)LSCs co-cultured onto TFs was significantly higher than those on DFs (p = 0.032) and 3T3-J2 (p = 0.047). Interestingly, LSCs co-cultures on TFs exhibited both significantly higher CFE and mRNA expression levels of ΔNp63α than on 3T3-J2 and DFs (p < 0.0001), showing also significantly greater levels of soluble factors (IL-6, HGF, b-FGF, G-CSF, TGF-β3) than LSCs on DFs. Therefore, TFs could represent an alternative feeder layer to both 3T3-J2 and DFs, potentially providing a suitable microenvironment for LSCs culture.

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.

Cardiac fibrosis and inflammation are major pathologic conditions that result from viral myocarditis. Connective tissue growth factor (CTGF) stimulates fibroblast proliferation and induces production of extracellular matrix molecules. We studied the correlation between CTGF and cardiac fibrosis in an acute Coxsackievirus B3 (CVB3) myocarditis animal model. Eight-week-old BALB/c mice were infected intraperitoneally with 10(4) plaque forming units (PFU) of CVB3. Myocardial inflammation peaked on day 7 and decreased markedly by day 14 post-infection (pi); cardiac fibrosis was noted from day 7 and peaked on day 14. By contrast, CTGF was weakly expressed by the interstitial cells in uninfected control hearts and also in the hearts of day 3 pi. CTGF expression measured by real-time PCR was elevated on day 3 and peaked on day 7 pi. TGF-beta expression peaked at day 7 pi. The cell type of CTGF expression changed from interstitial cells to myocytes after virus infection. On day 7, CTGF was strongly expressed by myocytes and inflammatory cells surrounding calcified necrotic areas. In addition, cardiac myocytes expressed CTGF on day 14. Our results, based on an acute CVB3 model of myocarditis, provide evidence that CTGF may mediate the development of fibrosis after viral myocarditis, and that the cells expressed CTGF changes during the course of viral myocarditis.

The fate of the palatal medial edge epithelial (MEE) cells undergoes programming cell death, migration, and epithelial-mesenchymal transdifferentiation (EMT) coincident with the process of palatal fusion and disappearance of MEE. Mesenchymal cells in the palate have both cranial neural crest (CNC) and non-CNC origins. The objectives of this study were to identify the populations of palatal mesenchymal cells using β-galactosidase (β-gal) and DiI cell lineage markers, and to determine whether MEE-derived cells continued to express transforming growth factor-β3 (TGF-β3) and transforming growth factor-β type III receptor (TβR-III), which were specific for MEE. A model has been developed using Wnt1 tissue specific expression of Cre-recombinase to activate β-gal solely in the CNC. The expressions of TGF-β3 and TβR-III in MEE were temporally correlated with critical events in palatogenesis. Three cell populations could be distinguished in the palatal mesenchymal CNC-derived, non-CNC derived and MEE-derived. After fusion, β-gal⁻ and DiI+ mesenchymal cells continued to express TGF-β3, however TβR-III was expressed only in the epithelial MEE, as well as keratin expression. In addition, we performed laser capture microdissection to identify mRNA expression of isolated DiI+ MEE cells. Both epithelial and transdifferentiated MEE have expressed TGF-β3, however, TβR-III was only expressed in epithelium. Extracellular matrix, especially MMP13 has been expressed coincident with fused stage which can be strongly associated with TGF-β3. These results demonstrate that combining a heritable marker and a cell lineage dye can distinguish different populations of mesenchymal cells in the developing palate. Furthermore, TGF-β3 and MMP13 could be strongly associated with EMT in palatogenesis.

BACKGROUND

Previous studies have indicated that transforming growth factor beta 3 (TGF-β3) was strongly expressed both in the gingival epithelium and the poorly structured pocket epithelium.

METHODS

A comprehensive analysis of the profile of tight junction proteins was carried out by quantitative real-time RT-PCR, Western blot and paracellular permeability assays.

RESULTS

Active TGF-β3 protein added to monolayers of cultured oral epithelial cells initially reduced the permeability to dextran (10 kDa), followed by an increase in permeability. Three hours after the addition of TGF-β3, expression of genes encoding tight junction components was selectively up- or down-regulated. In addition, up- or down-regulation of expression of several tight junction associated proteins was observed, although the protein changes did not parallel changes in gene expression. To confirm that TGF-β3 plays a role in epithelial barrier function, a selective Src family kinase inhibitor saracatinib (AZD0530) was added to cells treated with active TGF-β3. Tight junction proteins claudins-2, -20 and ZO-2 were significantly decreased, but claudin-4 and -18 were significantly increased.

CONCLUSIONS

These results suggest that TGF-β3 is involved in the modulation of epithelial barrier function by regulating assembly of tight junctions.