BACKGROUND

Pinocembrin is the most abundant flavonoid in propolis. In this study, we investigated the antimetastatic effect of pinocembrin on TGF-β1-induced epithelial-mesenchymal transition (EMT) and metastasis of human Y-79 retinoblastoma cells.

RESULTS

Firstly, the results showed that pinocembrin significantly suppresses the TGF-β1-induced abilities of the invasion and migration of Y-79 cells under non-cytotoxic concentration. Pinocembrin decreased TGF-β1-induced expression of vimentin, N-cadherin, αv and β3 integrin in Y-79 cells. Molecular data also showed pinocembrin inhibits the activation of focal adhesion kinase (FAK) and p38α signal involved in the downregulation of enzyme activities, protein and messenger RNA levels of matrix metalloproteinase-2/9 (MMP-2/-9) induced by TGF-β1. Next, pinocembrin also strongly inhibited the degradation of inhibitor of kappaBα (IκBα) and the nuclear levels of nuclear factor kappa B (NF-κB). Also, a dose-dependent inhibition on the binding ability of NF-κB was further observed under pinocembrin treatment.

CONCLUSIONS

Presented results indicated that pinocembrin inhibits TGF-β1-induced epithelial-mesenchymal transition (EMT) and metastasis of Y-79 cells by inactivating the αvβ3 integrin/FAK/p38α signaling pathway. Thus, our findings point to the anticancer potential of pinocembrin against retinoblastoma cells.

Damaged articular cartilage has limited self-healing capabilities, leading to degeneration that affects millions of people. Although cartilage tissue engineering is considered a promising approach for treatment, robust and long-term chondrogenesis within a 3-dimensional (3D) scaffold remains a major challenge for complete regeneration. Most current approaches involve incorporation of transforming growth factor-β (TGF-β) into the scaffold, but have limited utility owing to the short functional half-life and/or rapid clearance of TGF-β. In this study, we have tested the incorporation of graphene oxide nanosheets (GO) within a photopolymerizable poly-D, L-lactic acid/polyethylene glycol (PDLLA) hydrogel, for its applicability in sustained release of the chondroinductive growth factor TGF-β3. We found that with GO incorporation, the hydrogel scaffold (GO/PDLLA) exhibited enhanced initial mechanical strength, i.e., increased compressive modulus, and supported long-term, sustained release of TGF-β3 for up to 4 weeks. In addition, human bone marrow-derived mesenchymal stem cells (hBMSCs) seeded within TGF-β3 loaded GO/PDLLA hydrogels displayed high cell viability and improved chondrogenesis in a TGF-β3 concentration-dependent manner. hBMSCs cultured in GO/PDLLA also demonstrated significantly higher chondrogenic gene expression, including aggrecan, collagen type II and SOX9, and cartilage matrix production when compared to cultures maintained in GO-free scaffolds containing equivalent amounts of TGF-β3. Upon subcutaneous implantation in vivo, hBMSC-seeded TGF-β3-GO/PDLLA hydrogel constructs displayed considerably greater cartilage matrix than their TGF-β3/PDLLA counterparts without GO. Taken together, these findings support the potential application of GO in optimizing TGF-β3 induced hBMSC chondrogenesis for cartilage tissue engineering. Statement of Significance In this work, we have developed a graphene oxide (GO) incorporated, photocrosslinked PDLLA hybrid hydrogel for localized delivery and sustained release of loaded TGF-β3 to seeded cells. The incorporation of GO in PDLLA hydrogel suppressed the burst release of TGF-β3, and significantly prolonged the retention time of the TGF-β3 initially loaded in the hydrogel. Additionally, the GO improved the initial compressive strength of the hydrogel. Both in vitro analyses and in vivo implantation results showed that the GO/PDLLA constructs seeded with human mesenchymal stem cells (hMSCs) showed significantly higher cartilage formation, compared to GO-free scaffolds containing equivalent amount of TGF-β3. Findings from this work suggest the potential application of the GO-TGF/PDLLA hydrogel as a functional scaffold for hMSC-based cartilage tissue engineering.

The current study has investigated the use of decellularised, demineralised bone extracellular matrix (ECM) hydrogel constructs for in vivo tissue mineralisation and bone formation. Stro-1-enriched human bone marrow stromal cells were incorporated together with select growth factors including VEGF, TGF-β3, BMP-2, PTHrP and VitD3, to augment bone formation, and mixed with alginate for structural support. Growth factors were delivered through fast (non-osteogenic factors) and slow (osteogenic factors) release PLGA microparticles. Constructs of 5 mm length were implanted in vivo for 28 days within mice. Dense tissue assessed by micro-CT correlated with histologically assessed mineralised bone formation in all constructs. Exogenous growth factor addition did not enhance bone formation further compared to alginate/bone ECM (ALG/ECM) hydrogels alone. UV irradiation reduced bone formation through degradation of intrinsic growth factors within the bone ECM component and possibly also ECM cross-linking. BMP-2 and VitD3 rescued osteogenic induction. ALG/ECM hydrogels appeared highly osteoinductive and delivery of angiogenic or chondrogenic growth factors led to altered bone formation. All constructs demonstrated extensive host tissue invasion and vascularisation aiding integration and implant longevity. The proposed hydrogel system functioned without the need for growth factor incorporation or an exogenous inducible cell source. Optimal growth factor concentrations and spatiotemporal release profiles require further assessment, as the bone ECM component may suffer batch variability between donor materials. In summary, ALG/ECM hydrogels provide a versatile biomaterial scaffold for utilisation within regenerative medicine which may be tailored, ultimately, to form the tissue of choice through incorporation of select growth factors.

Implantation of recombinant human transforming growth factor-β3 (hTGF-β3) with coral-derived calcium carbonate-based macroporous bioreactors with limited conversion to hydroxyapatite (7% HA/CC) in the rectus abdominis muscle of the non-human primate Chacma baboon Papio ursinus induces endochondral bone formation. The exact mechanisms by which hTGF-β3 signalling induces bone in heterotopic sites of P. ursinus are not known. Coral-derived 7% HA/CC bioreactors with and without 125 μg hTGF-β3 were implanted in triplicate in the rectus abdominis muscle of 6 adult baboons. 7% HA/CC bioreactors either with or without hTGF-β3 were loaded with 125 μg of recombinant human Noggin (hNoggin), a bone morphogenetic proteins (BMPs) antagonist. Tissues on day 15, 60 and 90 were analysed by histomorphometry and quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR). Down-regulation of BMP-2 characterized 7% HA/CC constructs preloaded with 125 μg hNoggin with Noggin down-regulated on day 60 and 90 together with lack of TGF-β3 expression. Down-regulation of BMP-2 correlated with minimal bone formation by induction. hTGF-β3/hNoggin pre-treated bioreactors up-regulated BMP-2 but only on day 90 together with a significant down-regulation of Noggin on day 60 and 90, correlating with the induction of bone formation, albeit limited, on day 90 at the periphery of the macroporous bioreactors only. hTGF-β3 treated bioreactors significantly down-regulated BMP-2 on day 15 whilst up-regulating BMP-2 on day 60 and 90, together with down-regulation of Noggin on day 60 and 90 correlating with the prominent induction of bone formation. hTGF-β3 significantly up-regulated RUNX-2 and Osteocalcin expression on day 15 controlling the differentiation of progenitor stem cells into the osteoblastic lineage. The induction of bone as initiated by hTGF-β3 in the rectus abdominis muscle of P. ursinus is via the BMPs pathway with hTGF-β3 controlling the induction of bone formation by regulating the expression of BMPs via Noggin expression. These results unequivocally demonstrate that hTGF-β3 elicits bone induction by up-regulation of endogenous BMP-2 and is blocked by hNoggin.

TGF-β isoforms (TGF-β1, -β2, and -β3) are secreted signaling ligands that stimulate the expression of protein components of the extracellular matrix, regulate the growth and differentiation of epithelial cells, modulate immune cell function, and play roles in the development of several essential organs, including the heart and lungs. The importance of the TGF-βs is underscored by their conservation among vertebrates and by their demonstrated roles in a variety of human diseases, including tissue fibrosis and cancer. The objective of this review is to highlight recent progress in characterizing the structures of the three TGF-β isoforms in complex with their receptors, and to compare these with one another as well as with other members of the superfamily. Although the structural information and accompanying biophysical studies emphasize the shared ancestry of TGF-βs, they also provide insight as to how the TGF-βs diverged from other members of the superfamily and one another to fulfill distinct roles in vivo. The similarities and differences by which the isoforms bind their receptors present unique opportunities for designing pan-isoform and isoform-specific ligand traps and progress toward developing these is described.

Systemic lupus erythematosus (SLE) is a prototypical autoimmune disease characterized by multiorgan inflammation induced by autoantibodies. Early growth response gene 2 (Egr2), a transcription factor essential for T-cell anergy induction, controls systemic autoimmunity in mice and humans. We have previously identified a subpopulation of CD4+ regulatory T cells, CD4+CD25-LAG3+ cells, that characteristically express both Egr2 and LAG3 and control mice model of lupus via TGF-β3 production. However, due to the mild phenotype of lymphocyte-specific Egr2-deficient mice, the presence of an additional regulator has been speculated. Here, we show that Egr2 and Egr3 expressed in T cells cooperatively prevent humoral immune responses by supporting TGF-β3 secretion. T cell-specific Egr2/Egr3 double-deficient (Egr2/3DKO) mice spontaneously developed an early onset lupus-like disease that was more severe than in T cell-specific Egr2-deficient mice. In accordance with the observation that CD4+CD25-LAG3+ cells from Egr2/3DKO mice completely lost the capacity to produce TGF-β3, the excessive germinal center reaction in Egr2/3DKO mice was suppressed by the adoptive transfer of WT CD4+CD25-LAG3+ cells or treatment with a TGF-β3-expressing vector. Intriguingly, latent TGF-β binding protein (Ltbp)3 expression maintained by Egr2 and Egr3 was required for TGF-β3 production from CD4+CD25-LAG3+ cells. Because Egr2 and Egr3 did not demonstrate cell intrinsic suppression of the development of follicular helper T cells, Egr2- and Egr3-dependent TGF-β3 production by CD4+CD25-LAG3+ cells is critical for controlling excessive B-cell responses. The unique attributes of Egr2/Egr3 in T cells may provide an opportunity for developing novel therapeutics for autoantibody-mediated diseases including SLE.

Burn wounds are associated with a series of risks, such as infection and pathologic scar tissue formation, which significantly delay wound healing and lead to complications. In this study, we successfully fabricated a dextran-hyaluronic acid (Dex-HA) hydrogel enriched with sanguinarine (SA) incorporated into gelatin microspheres (GMs), which had high porosity, good swelling ratio, enhanced NIH-3T3 fibroblast cell proliferation, and sustained SA release profile. The in vitro degradation results indicate that the SA/GMs/Dex-HA hydrogel can be degraded. The in vitro antibacterial tests showed that the SA/GMs/Dex-HA hydrogel can inhibit methicillin-resistant Staphylococcus aureus (MRSA) and Escherichia coli (E. coli). We evaluated the wound-healing effects and antibacterial properties of SA/GMs/Dex-HA hydrogels in a rat full-thickness burn infection model. The hematoxylin-eosin (H&E) and Masson's trichrome staining results of the SA/GMs/Dex-HA hydrogel showed that it improved re-epithelialization and enhanced extracellular matrix remodeling, and immunohistochemistry results showed that the expression of TGF-β1 and TNF-α was decreased, while the TGF-β3 expression was increased. Our findings suggest that the SA/GMs/Dex-HA hydrogel provides a potential way for infected burn treatment with high-quality and efficient scar inhibition.

Stem cells (SCs) are a promising approach to regenerative medicine, with the potential to treat numerous orthopedic disorders, including osteo-degenerative diseases. The development of human-induced pluripotent stem cells (hiPSCs) has increased the potential of SCs for new treatments. However, current methods of differentiating hiPSCs into chondrocyte-like cells are suboptimal and better methods are needed. The aim of the present study was to assess four different chondrogenic differentiation protocols to identify the most efficient method of generating hiPSC-derived chondrocytes. For this study, hiPSCs were obtained from primary hu man dermal fibroblasts (PHDFs) and differentiated into chondrocyte-like cells using four different protocols: 1) monolayer culture with defined growth factors (GF); 2) embryoid bodies (EBs) in a chondrogenic medium with TGF-β3 cells; 3) EBs in chondrogenic medium conditioned with human chondrocytes (HC-402-05a cell line) and 4) EBs in chondrogenic medium conditioned with human chondrocytes and supplemented with TGF-β3. The cells obtained through these four protocols were evaluated and compared at the mRNA and protein levels. Although chondrogenic differentiation of hiPSCs was successfully achieved with all of these protocols, the two fastest and most cost-effective methods were the monolayer culture with GFs and the medium conditioned with human chondrocytes. Both of these methods are superior to other available techniques. The main advantage of the conditioned medium is that the technique is relatively simple and inexpensive while the directed method (i.e., monolayer culture with GFs) is faster than any protocol described to date because it is does not require additional steps such as EB formation.

The adhesion of cardiac fibroblasts to the glycated collagen interstitium in diabetics is associated with de novo expression of the α11 integrin, myofibroblast formation and cardiac fibrosis. We examined how methylglyoxal-glycated collagen regulates α11 integrin expression. In cardiac fibroblasts plated on glycated collagen but not glycated fibronectin, there was markedly increased α11 integrin and α-smooth muscle actin expression. Compared with native collagen, binding of purified α11β1 integrin to glycated collagen was reduced by>>fourfold, which was consistent with reduced fibroblast attachment to glycated collagen. Glycated collagen strongly enhanced the expression of TGF-β2 but not TGF-β1 or TGF-β3. The increased expression of TGF-β2 was inhibited by triple helical collagen peptides that mimic the α11β1 integrin binding site on type I collagen. In cardiac fibroblasts transfected with α11 integrin luciferase promoter constructs, glycated collagen activated the α11 integrin promoter. Analysis of α11 integrin promoter truncation mutants showed a novel Smad2/3 binding site located between -809 and -1300 nt that was required for promoter activation. We conclude that glycated collagen in the cardiac interstitium triggers an autocrine TGF-β2 signaling pathway that stimulates α11 integrin expression through Smad2/3 binding elements in the α11 integrin promoter, which is important for myofibroblast formation and fibrosis.

Butyric acid is a major short chain fatty acid (SCFA), produced in the gastrointestinal tract by anaerobic bacterial fermentation, that has beneficial health effects in many species including poultry. To understand the immunomodulating effects of butyrate on avian macrophage, we treated a naturally transformed line of chicken macrophage cells named HTC with Na-butyrate in the absence or presence of Salmonella typhimurium lipopolysaccharide (LPS) or phorbol-12-myristate-13-acetate (PMA), a metabolic activator, evaluating its various functional parameters. The results demonstrate that, butyrate by itself had no significant effect on variables such as nitric oxide (NO) production and the expression of genes associated with various inflammatory cytokines but it inhibited NO production, and reduced the expression of cytokines such as IL-1β, IL-6, IFN-γ, and IL-10 in LPS-stimulated cells. Butyrate decreased the expression of TGF-β3 in the presence or absence of LPS, while it had no effect on IL-4, Tβ4, and MMP2 gene expression. In addition, butyrate augmented PMA induced oxidative burst indicated by DCF-DA oxidation and restored LPS induced attenuation of tartrate resistant acid phosphatase (TRAP) activity. Although butyrate had no significant effect on phagocytosis or matrix metalloproteinase (MMP) activities of resting macrophages, it significantly suppressed the effects induced by their respective stimulants such as LPS induced phagocytosis and PMA induced MMP expression. These results suggest that butyrate has immunomodulatory property in the presence of agents that incite the cells thus, has potential to control inflammation and restore immune homeostasis.

Periodontitis is a local inflammatory disease initiated by bacteria accumulation and results in cytokine mediated alveolar bone resorption and tissue destruction. In this study, the effect of locally delivered atorvastatin (2% w/v) containing chitosan formulations in the treatment of periodontitis was evaluated in rats with ligature induced periodontitis. The levels of interleukin-1beta (IL-1β), IL-6, IL-8, IL-10, transforming growth factor-β1 (TGF-β1), TGF-β2 and TGF-β3 were measured after treatment with formulations. Histomorphometric analysis included the measurements of the area of alveolar bone and the distance between cemento-enamel junction (CEJ) and connective tissue attachment to tooth. Inflammatory and osteoclastic activity scores were given semiquantitatively. Following the administration of atorvastatin, release of pro-inflammatory (IL-1β, IL-6 and IL-8) and anti-inflammatory (TGF-β1 and TGF-β2) cytokines was found to decrease, with a significant alveolar bone healing, when compared to that of control. The anti-inflammatory effect was observed to enhance in presence of chitosan. These findings suggest that chitosan based delivery system for a statin group drug, atorvastatin is a promising for the treatment of periodontal disease.

Focal articular cartilage (AC) defects, if left untreated, can lead to debilitating diseases such as osteoarthritis. While several tissue engineering strategies have been developed to promote cartilage regeneration, it is still challenging to generate functional AC capable of sustaining high load-bearing environments. Here, a new class of cartilage extracellular matrix (cECM)-functionalized alginate bioink is developed for the bioprinting of cartilaginous tissues. The bioinks are 3D-printable, support mesenchymal stem cell (MSC) viability postprinting and robust chondrogenesis in vitro, with the highest levels of COLLII and ACAN expression observed in bioinks containing the highest concentration of cECM. Enhanced chondrogenesis in cECM-functionalized bioinks is also associated with progression along an endochondral-like pathway, as evident by increases in RUNX2 expression and calcium deposition in vitro. The bioinks loaded with MSCs and TGF-β3 are also found capable of supporting robust chondrogenesis, opening the possibility of using such bioinks for direct "print-and-implant" cartilage repair strategies. Finally, it is demonstrated that networks of 3D-printed polycaprolactone fibers with compressive modulus comparable to native AC can be used to mechanically reinforce these bioinks, with no loss in cell viability. It is envisioned that combinations of such biomaterials can be used in multiple-tool biofabrication strategies for the bioprinting of biomimetic cartilaginous implants.

Patients with cervical cancer (CxCa) typically present an infiltrate of tumor-associated macrophages, which is associated with a poor prognosis. We found that CxCa cell lines (HeLa, SiHa, and C-33A) secreted factors involved in regulating tumor growth including IL-6, IL-4, PDGFAA, HGF, VEGF, ANG-2, and TGF-β3. We assessed the effects of culture supernatants from these cell lines on macrophages derived from the THP-1 cell line. Macrophages treated with culture supernatants from CxCa cells developed an M2-like phenotype with expression of CD163, low nitric oxide release, and high secretion of IL-6, PDGFAA, HGF, ANG-2, and VEGF. The macrophages continued to produce PDGFAA, PDGFBB, and VEGF 48h after the CxCa cell culture supernatants were removed. The induction of M2 macrophages in vivo favors tumor growth, angiogenesis, tissue remodeling, and metastasis. These results demonstrated that factors secreted by CxCa cells induced a stable M2 phenotype in THP-1 macrophages.

The aqueous humor (AH) component transforming growth factor (TGF)-β2 is strongly correlated to primary open-angle glaucoma (POAG), and was shown to up-regulate glaucoma-associated extracellular matrix (ECM) components, members of the ECM degradation system and heat shock proteins (HSP) in primary ocular cells. Here we present osteopontin (OPN) as a new TGF-β2 responsive factor in cultured human optic nerve head (ONH) astrocytes. Activation was initially demonstrated by Oligo GEArray microarray and confirmed by semiquantitative (sq) RT-PCR, realtime RT-PCR and western blot. Expressions of most prevalent OPN receptors CD44 and integrin receptor subunits αV, α4, α 5, α6, α9, β1, β3 and β5 by ONH astrocytes were shown by sqRT-PCR and immunofluorescence labeling. TGF-β2 treatment did not affect their expression levels. OPN did not regulate gene expression of described TGF-β2 targets shown by sqRT-PCR. In MTS-assays, OPN had a time- and dose-dependent stimulating effect on the metabolic activity of ONH astrocytes, whereas TGF-β2 significantly reduced metabolism. OPN signaling via CD44 mediated a repressive outcome on metabolic activity, whereas signaling via integrin receptors resulted in a pro-metabolic effect. In summary, our findings characterize OPN as a TGF-β2 responsive factor that is not involved in TGF-β2 mediated ECM and HSP modulation, but affects the metabolic activity of astrocytes. A potential involvement in a protective response to TGF-β2 triggered damage is indicated, but requires further investigation.

The roles of long noncoding RNAs (lncRNAs) in musculoskeletal development, disease, and regeneration remain poorly understood. Here, we identified the novel lncRNA GRASLND (originally named RNF144A-AS1) as a regulator of mesenchymal stem cell (MSC) chondrogenesis. GRASLND, a primate-specific lncRNA, is upregulated during MSC chondrogenesis and appears to act directly downstream of SOX9, but not TGF-b3. We showed that the silencing of GRASLND resulted in lower accumulation of cartilage-like extracellular matrix in a pellet assay, while GRASLND overexpression - either via transgene ectopic expression or by endogenous activation via CRISPR-dCas9-VP64 - significantly enhanced cartilage matrix production. GRASLND acts to inhibit IFN-γ by binding to EIF2AK2, and we further demonstrated that GRASLND exhibits a protective effect in engineered cartilage against interferon type II. Our results indicate an important role of GRASLND in regulating stem cell chondrogenesis, as well as its therapeutic potential in the treatment of cartilage-related diseases, such as osteoarthritis.

BACKGROUND

Chondrogenic differentiation of mesenchymal stem cells (MSCs) is important for osteoarthritis (OA) treatment. However, the specific mechanisms involved are undefined. MicroRNAs (miRNAs) downregulate protein synthesis by binding to the 3'UTR of target mRNA.

METHODS

Bone marrow aspirates were obtained from OA patients undergoing total hip arthroplasty (n=8) to isolate MSCs. MiR-410 or miR-410 inhibitor were transfected into MSCs using lentivirus and the effects were assessed. Alcian blue staining detected differences in chondrogenic differentiation. An MTT assay and flow cytometry determined changes in cell proliferation and cell cycle, respectively. Real time PCR assessed differences in miRNA and mRNA expression levels and western blotting detected changes in protein levels. ChIP assessed differences in transcriptional activation. TOP/FOP determined changes in the activity of the Wnt signaling pathway. A dual-luciferase reporter assay was used to confirm the miR-410 target protein.

RESULTS

miR-410 was elevated during transforming growth factor β3 (TGF-β3)-induced chondrogenic differentiation of MSCs. miR-410 targeted a putative binding site in the 3'-UTR of the Wnt3a gene, thus regulating the Wnt signaling pathway. miR-410 transfection increased mRNA and protein levels of four chondrogenic markers, type II collagen (Col2a1), SRY-box 9 (Sox9), aggrecan (ACAN), and hyaluronan synthase 2 (Has2). miR-410 overexpression decreased Wnt3a protein expression. Wnt3a levels increased in OA patient cartilage concomitant with OA severity and significantly negatively correlated with miR-410 levels.

CONCLUSIONS

miR-410 is a key regulator of MSC chondrogenic differentiation and directly targets Wnt3a triggering the Wnt signaling pathway.

Adipose-derived stromal vascular fraction (SVF) is a heterogeneous population of cells that yields a homogeneous population of plastic-adherent adipose tissue-derived stromal cells (ASC) when culture-expanded. SVF and ASC have been used clinically to improve tendon healing, yet their mechanism of action is not fully elucidated. The objective of this study was to investigate the potential for ASC to act as trophic mediators for tendon healing. Flexor digitorum superficialis tendons and adipose tissue were harvested from adult horses to obtain SVF, ASC, and tenocytes. Growth factor gene expression was quantified in SVF and ASC in serial passages and growth factors were quantified in ASC-conditioned medium (CM). Microchemotaxis assays were performed using ASC-CM. Tenocytes were grown in co-culture with autologous ASC or allogeneic SVF. Gene expression for insulin-like growth factor 1 (IGF-1), stromal cell-derived factor-1α (SDF-1α), transforming growth factor-β1 (TGF-β1) and TGF-β3 was significantly higher in SVF compared to ASC. Concentrations were significantly increased in ASC-CM compared to controls for IGF-1 (4-fold) and SDF-1α (6-fold). Medium conditioned by ASC induced significant cell migration in a dose-dependent manner. Gene expression for collagen types I and III, decorin, and cartilage oligomeric matrix protein was modestly, but significantly increased following co-culture of tenocytes with autologous ASC. Our findings support the ability of SVF and ASC to act as trophic mediators in tendon healing, particularly through chemotaxis, which stands to critically impact the intrinsic healing response. In vivo studies to further delineate the potential for SVF and/or ASC to improve tendon healing are warranted. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:1429-1439, 2019.

OBJECTIVE

The transcriptomes of seven diverse clonal human embryonic progenitor cell lines with chondrogenic potential were compared with that of bone marrow-derived mesenchymal stem cells (MSCs).

METHODS

The cell lines 4D20.8, 7PEND24, 7SMOO32, E15, MEL2, SK11 and SM30 were compared with MSCs using immunohistochemical methods, gene expression microarrays and quantitative real-time PCR.

RESULTS

In the undifferentiated progenitor state, each line displayed unique combinations of site-specific markers, including AJAP1, ALDH1A2, BMP5, BARX1, HAND2, HOXB2, LHX1, LHX8, PITX1, TBX15 and ZIC2, but none of the lines expressed the MSC marker CD74. The lines showed diverse responses when differentiated in the presence of combinations of TGF-β3, BMP2, 4, 6 and 7 and GDF5, with the lines 4D20.8, SK11, SM30 and MEL2 showing osteogenic markers in some differentiation conditions. The line 7PEND24 showed evidence of regenerating articular cartilage and, in some conditions, markers of tendon differentiation.

CONCLUSIONS

The scalability of site-specific clonal human embryonic stem cell-derived embryonic progenitor cell lines may provide novel models for the study of differentiation and methods for preparing purified and identified cells types for use in therapy.

Overexpression of the nuclear receptor 4A1 (NR4A1) in breast cancer patients is a prognostic factor for decreased survival and increased metastasis, and this has been linked to NR4A1-dependent regulation of transforming growth factor β (TGF-β) signaling. Results of RNA interference studies demonstrate that basal migration of aggressive SKBR3 and MDA-MB-231 breast cancer cells is TGF-β independent and dependent on regulation of β1-integrin gene expression by NR4A1 which can be inhibited by the NR4A1 antagonists 1,1-bis(3'-indolyl)-1-(p-hydroxyphenyl)methane (DIM-C-pPhOH) and a related p-carboxymethylphenyl [1,1-bis(3'-indolyl)-1-(p-carboxymethylphenyl)methane (DIM-C-pPhCO2Me)] analog. The NR4A1 antagonists also inhibited TGF-β-induced migration of MDA-MB-231 cells by blocking nuclear export of NR4A1, which is an essential step in TGF-β-induced cell migration. We also observed that NR4A1 regulates expression of both β1- and β3-integrins, and unlike other β1-integrin inhibitors which induce prometastatic β3-integrin, NR4A1 antagonists inhibit expression of both β1- and β3-integrin, demonstrating a novel mechanism-based approach for targeting integrins and integrin-dependent breast cancer metastasis.

Tendon adhesion is a common problem in the healing of injured tendons. The molecular mechanisms of the TGF-β/Smad signaling pathway have been determined, and the role of TGF-β has been well characterized in wound healing. However, the intracellular mechanism or downstream signals by which TGF-β3 modulates its effects on tendon healing have not been well elucidated. The aim of this study was to determine the effect of TGF‑β3 on the TGF-β/Smad signaling pathway in tenocytes. Quantitative polymerase chain reaction and western blot analysis were used to analyze the effect of TGF‑β3 on the regulation of the expression of Smad proteins in tenocytes. The results demonstrated that TGF‑β3 has no significant effect on the proliferation of tendon cells. The addition of TGF‑β3 to tenocytes can significantly downregulate the expression of Smad3 and upregulate the expression of Smad7 at the gene and protein levels. The results demonstrate that TGF‑β3 may regulate Smad3 and Smad7 proteins through the TGF-β/Smad signaling pathway to minimize extrinsic scarring. Thus, it may provide a novel approach to decrease tendon adhesion and promote tendon healing.

BACKGROUND

Esophageal cancer is often marked by aggressive tumor growth and poor prognosis. Patient groups who benefit from perioperative therapy are not yet defined. The tumor microenvironment and circulating factors as possible predictors of response and prognosis gain interest. This study aimed to investigate cytokines in patients' serum and tumor tissue with regard to response and prognosis.

RESULTS

Median survival between SCC and AC was not different (published previously). Lower levels of CCL11 (Eotaxin-1) and CXCL10 (IP-10) in the tumor tissue were associated with a better prognosis (p = 0.022; p = 0.002). In the AC subgroup higher concentrations of TGF-β3 in serum and corresponding tumor tissue were associated with adverse prognosis (p = 0.035; p = 0.006). An association with histopathological response was found for IL-12(p70) and CXCL10 in patients' sera (p = 0.041; p = 0.032). The tissue levels of TGF-β1 and TGF-β2 were significantly lower in histopathological responders than in nonresponders (p = 0.033; p = 0.007). A similar trend was seen for TGF-β3, without statistical significance (p = 0.097).

METHODS

Preoperative serum samples and corresponding tumor tissue (n = 54), only serum (n = 20) or only tissue (n = 4) were collected from patients undergoing surgery for cT3/4 esophageal squamous cell cancer (SCC) (n = 34) and adenocarcinoma (AC) (n = 44). All samples were taken after neoadjuvant treatment. All patients received perioperative chemo(radio)therapy. Cytokine levels of 17 different cytokines were measured by multiplex immunoassay and correlated with clinicopathological factors.

CONCLUSIONS

Two chemokines (CCL11 and CXCL10) in posttherapeutic tumor tissue were associated with prognosis in patients with esophageal cancer, lower levels indicating a better prognosis. Lower levels of TGF-β were associated with better response and prognosis in patients with AC.

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.

Hydrogel precursors are liquid solutions that are prone to leaking after surgical placement. This problem was overcome by incorporating either decellularized cartilage (DCC) or devitalized cartilage (DVC) microparticles into traditional photocrosslinkable hydrogel precursors in an effort to achieve a paste-like hydrogel precursor. DCC and DVC were selected specifically for their potential to induce chondrogenesis of stem cells, given that materials that are chondroinductive on their own without growth factors are a revolutionary goal in orthopedic medicine. We hypothesized that DVC, lacking the additional chemical processing steps in DCC to remove cell content, would lead to a more chondroinductive hydrogel with rat bone marrow-derived mesenchymal stem cells. Hydrogels composed of methacrylated hyaluronic acid (MeHA) and either DCC or DVC microparticles were tested with and without exposure to transforming growth factor (TGF)-β3 over a 6 week culture period, where swelling, mechanical analysis, and gene expression were observed. For collagen II, Sox-9, and aggrecan expression, MeHA precursors containing DVC consistently outperformed the DCC-containing groups, even when the DCC groups were exposed to TGF-β3. DVC consistently outperformed all TGF-β3-exposed groups in aggrecan and collagen II gene expression as well. In addition, when the same concentrations of MeHA with DCC or DVC microparticles were evaluated for yield stress, the yield stress with the DVC microparticles was 2.7 times greater. Furthermore, the only MeHA-containing group that exhibited shape retention was the group containing DVC microparticles. DVC appeared to be superior to DCC in both chondroinductivity and rheological performance of hydrogel precursors, and therefore DVC microparticles may hold translational potential for cartilage regeneration.