Enhanced chondrogenic potential in GelMA-based 3D cartilage model via Wnt3a surface immobilization.

Cartilage tissue engineering aims to develop functional substitutes for treating cartilage defects and osteoarthritis. Traditional two-dimensional (2D) cell culture systems lack the complexity of native cartilage, leading to the development of 3D regenerative cartilage models. In this study, we developed a 3D model using Gelatin Methacryloyl (GelMA)-based hydrogels seeded with Y201 cells, a bone marrow mesenchymal stem cell line. The model investigated chondrogenic differentiation potential in response to Wnt3a stimulation within the GelMA scaffold and validated using known chondrogenic agonists. Y201 cells demonstrated suitability for the model, with increased proteoglycan content and upregulated chondrogenic marker expression under chondrogenic conditions. Wnt3a enhanced cell proliferation, indicating activation of the Wnt/ß-catenin pathway, which plays a role in cartilage development. GelMA hydrogels provided an optimal scaffold, supporting cell viability and proliferation. The 3D model exhibited consistent responses to chondrogenic agonists, with TGF-ß3 enhancing cartilage-specific extracellular matrix (ECM) production and chondrogenic differentiation. The combination of Wnt3a and TGF-ß3 showed synergistic effects, promoting chondrogenic differentiation and ECM production. This study presents a 3D regenerative cartilage model with potential for investigating cartilage biology, disease mechanisms, and drug screening. The model provides insights into complex cartilage regeneration mechanisms and offers a platform for developing therapeutic approaches for cartilage repair and osteoarthritis treatment.

Characterizing soluble immune checkpoint molecules and TGF-ß1,2,3 in pleural effusion of malignant pleural mesothelioma.

The clinical impact of soluble molecules in pleural effusion (PE) is unclear in patients with malignant pleural mesothelioma (MPM). In this single-center, retrospective, observational study, we assessed soluble forms of cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), programmed cell death protein 1 (PD-1), and PD-1 ligand 1 (PD-L1) using enzyme-linked immunosorbent assays; three TGF-ß isoforms were measured via multiplex assay in PE of patients with fibrinous pleuritis (FP) or MPM, to assess relationships between the levels of six molecules, clinicopathological characteristics, and efficacy of immune checkpoint inhibitors. Soluble forms of CTLA-4, PD-L1, PD-1, TGF-ß1, TGF-ß2, and TGF-ß3 were variably produced in PE of FP (n = 34) and MPM (n = 79); we found significant relationships between the six molecules and clinicopathological features. Although none of the three soluble immune checkpoint molecules showed diagnostic or prognostic effects in patients with MPM, TGF-ß2 level in PE is a useful differential diagnostic marker between FP and MPM. Both TGF-ß1 and TGF-ß3 levels are promising prognostic markers for MPM. Moreover, we found that higher baseline levels of PD-1 soluble forms predicted the response to anti-PD1 monotherapy. Our findings identify novel diagnostic, prognostic, and predictive biomarkers for anti-PD1 therapy in patients with MPM.

Genome-wide in vivo CRISPR screen identifies TGFß3 as actionable biomarker of palbociclib resistance in triple negative breast cancer.

Triple negative breast cancer (TNBC) remains exceptionally challenging to treat. While CDK4/6 inhibitors have revolutionized HR + breast cancer therapy, there is limited understanding of their efficacy in TNBC and meaningful predictors of response and resistance to these drugs remain scarce. We conducted an in vivo genome-wide CRISPR screen using palbociclib as a selection pressure in TNBC. Hits were prioritized using microarray data from a large panel of breast cancer cell lines to identify top palbociclib sensitizers. Our study defines TGFß3 as an actionable determinant of palbociclib sensitivity that potentiates its anti-tumor effects. Mechanistically, we show that chronic palbociclib exposure depletes p21 levels, contributing to acquired resistance, and that TGFß3 treatment can overcome this. This study defines TGFß3 as an actionable biomarker that can be used to improve patient stratification for palbociclib treatment and exploits the synergistic interaction between CDK4/6 and TGFß3 to propose a new combinatorial treatment for TNBC.

Differential Smad2/3 linker phosphorylation is a crosstalk mechanism of Rho/ROCK and canonical TGF-ß3 signaling in tenogenic differentiation.

The transforming growth factor (TGF)-ß3 is a well-known inducer for tenogenic differentiation, signaling via the Smad2/3 pathway. Furthermore, other factors like extracellular matrix or mechanical force can induce tenogenic differentiation and possibly alter the response to TGF-ß3 by signaling via the Rho/ROCK pathway. The aim of this study was to investigate the interplay of Rho/ROCK and TGF-ß3/Smad signaling in tenogenic differentiation, with the Smad2/3 molecule hypothesized as a possible interface. Cultured as monolayers or on collagen I matrices, mesenchymal stromal cells (MSC) were treated with the ROCK inhibitor Y-27632 (10 µM), TGF-ß3 (10 ng/ml) or both combined. Control cells were cultured accordingly, without Y-27632 and/or without TGF-ß3. At different time points, MSC were analyzed by real-time RT-PCR, immunofluorescence, and Western blot. Cultivation of MSC on collagen matrices and ROCK inhibition supported tenogenic differentiation and fostered the effect of TGF-ß3. The phosphorylation of the linker region of Smad2 was reduced by cultivation on collagen matrices, but not by ROCK inhibition. The latter, however, led to increased phosphorylation of the linker region of Smad3. In conclusion, collagen matrices and the Rho/ROCK signaling pathway influence the TGF-ß3/Smad2/3 pathway by regulating different phosphorylation sites of the Smad linker region.

Evaluation of differences in expression pattern of three isoforms of the transforming growth factor beta in patients with lumbosacral stenosis.

The study investigates molecular changes in the lumbosacral (L/S) spine's yellow ligamentum flavum during degenerative stenosis, focusing on the role of transforming growth factor beta 1-3 (TGF-ß-1-3). Sixty patients with degenerative stenosis and sixty control participants underwent molecular analysis using real-time quantitative reverse transcription reaction technique (RTqPCR), enzyme-linked immunosorbent assay (ELISA), Western blot, and immunohistochemical analysis (IHC). At the mRNA level, study samples showed reduced expression of TGF-ß-1 and TGF-ß-3, while TGF-ß-2 increased by only 4%. Conversely, at the protein level, the study group exhibited significantly higher concentrations of TGF-ß-1, TGF-ß-2, and TGF-ß-3 compared to controls. On the other hand, at the protein level, a statistically significant higher concentration of TGF-ß-1 was observed (2139.33 pg/mL ± 2593.72 pg/mL vs. 252.45 pg/mL ± 83.89 pg/mL; p < 0.0001), TGF-ß-2 (3104.34 pg/mL ± 1192.74 pg/mL vs. 258.86 pg/mL ± 82.98 pg/mL; p < 0.0001), TGF-ß-3 (512.75 pg/mL ± 107.36 pg/mL vs. 55.06 pg/mL ± 9.83 pg/mL, p < 0.0001) in yellow ligaments obtained from patients of the study group compared to control samples. The study did not establish a significant correlation between TGF-ß-1-3 concentrations and pain severity. The findings suggest that molecular therapy aimed at restoring the normal expression pattern of TGF-ß-1-3 could be a promising strategy for treating degenerative stenosis of the L/S spine. The study underscores the potential therapeutic significance of addressing molecular changes at the TGF-ß isoforms level for better understanding and managing degenerative spinal conditions.

TGF-ß3 from fibroblasts promotes necrotising sialometaplasia by suppressing salivary gland cell proliferation and inducing squamous metaplasia.

Necrotising sialometaplasia (NSM) is a non-neoplastic lesion mainly arising in the minor salivary glands of the oral cavity. In the clinical features, NSM shows swelling with or without ulceration, and can mimic a malignant disease such as squamous cell carcinoma. Histopathologically, NSM usually shows the lobular architecture that is observed in the salivary glands. Additionally, acinar infarction and squamous metaplasia of salivary ducts and acini are observable. The aetiology of this lesion remains unknown, although it has a characteristic feature that sometimes requires clinical and histopathological differentiation from malignancy. In this study, we investigated upregulated genes in NSM compared with normal salivary glands, and focused on the TGF-ß3 (TGFB3) gene. The results of the histopathological studies clarified that fibroblasts surrounding the lesion express TGF-ß3. Moreover, in vitro studies using mouse salivary gland organoids revealed that TGF-ß3 suppressed salivary gland cell proliferation and induced squamous metaplasia. We demonstrated a possible aetiology of NSM by concluding that increased TGF-ß3 expression during wound healing or tissue regeneration played a critical role in cell proliferation and metaplasia. © 2024 The Pathological Society of Great Britain and Ireland.

Effect of simultaneous and sequential use of TGF-ß1 and TGF-ß3 with FGF-2 on teno/ligamentogenic differentiation of periodontal ligament stem cells.

OBJECTIVE: The periodontal ligament is a crucial part of the periodontium, and its regeneration is challenging. This study compares the effect of simultaneous and sequential use of FGF-2 and TGF-ß1 with FGF-2 and TGF-ß3 on the periodontal ligament stem cells (PDLSCs) teno/ligamentogenic differentiation. DESIGN: This study comprises ten different groups. A control group with only PDLSCs; FGF-2 group containing PDLSCs with a medium culture supplemented with FGF-2 (50 ng/mL). In other experimental groups, different concentrations (5 ng/mL or 10 ng/mL) of TGF-ß1&-ß3 simultaneously or sequentially were combined with FGF-2 on the cultured PDLSCs. TGF-ß was added to the medium after day 3 in the sequential groups. Methyl Thiazolyl Tetrazolium (MTT) assay on days 3, 5, and 7 and Quantitative Real-time Polymerase Chain Reaction (RT-qPCR) analysis after day 7 were conducted to investigate PLAP1, SCX, and COL3A1, RUNX2 genes. All experiments were conducted in a triplicate. The One-way and Two-way ANOVA with Tukey post hoc were utilized to analyze the results of the MTT and RT-qPCR tests, respectively. A p-value less than 0.05 is considered significant. RESULTS: The proliferation of cells on days 3, 5, and 7 was not significantly different among different experimental groups (P > 0.05). A higher expression of the PLAP1, SCX, and COL3A1 have been seen in groups with sequential use of growth factors; among these groups, the group using 5 ng/mL of TGF-ß3 led other groups with the most amount of significant upregulation in PLAP1(17.69 ± 1.11 fold; P < 0.0001), SCX (5.71 ± 0.38 fold; P < 0.0001), and COL1A3 (6.35 ± 0.39 fold; P < 0.0001) expression, compared to the control group. The expression of the RUNX2 decreased in all groups compared to the control group; this reduction was more in groups with sequential use of growth factors. CONCLUSION: The sequential use of growth factors can be more effective than simultaneous use in teno/ligamentogenic differentiation of PDLSCs. Moreover, treatment with 5 ng/mL TGF-ß3 after FGF-2 was more effective than TGF-ß1.

TGF-ß3 increases the severity of radiation-induced oral mucositis and salivary gland fibrosis in a mouse model.

PURPOSE: Toxicities from head and neck (H&N) radiotherapy (RT) may affect patient quality of life and can be dose-limiting. Proteins from the transforming growth factor beta (TGF-ß) family are key players in the fibrotic response. While TGF-ß1 is known to be pro-fibrotic, TGF-ß3 has mainly been considered anti-fibrotic. Moreover, TGF-ß3 has been shown to act protective against acute toxicities after radio- and chemotherapy. In the present study, we investigated the effect of TGF-ß3 treatment during fractionated H&N RT in a mouse model. MATERIALS AND METHODS: 30 C57BL/6J mice were assigned to three treatment groups. The RT + TGF-ß3 group received local fractionated H&N RT with 66 Gy over five days, combined with TGF-ß3-injections at 24-hour intervals. Animals in the RT reference group received identical RT without TGF-ß3 treatment. The non-irradiated control group was sham-irradiated according to the same RT schedule. In the follow-up period, body weight and symptoms of oral mucositis and lip dermatitis were monitored. Saliva was sampled at five time points. The experiment was terminated 105 d after the first RT fraction. Submandibular and sublingual glands were preserved, sectioned, and stained with Masson's trichrome to visualize collagen. RESULTS: A subset of mice in the RT + TGF-ß3 group displayed increased severity of oral mucositis and increased weight loss, resulting in a significant increase in mortality. Collagen content was significantly increased in the submandibular and sublingual glands for the surviving RT + TGF-ß3 mice, compared with non-irradiated controls. In the RT reference group, collagen content was significantly increased in the submandibular gland only. Both RT groups displayed lower saliva production after treatment compared to controls. TGF-ß3 treatment did not impact saliva production. CONCLUSIONS: When repeatedly administered during fractionated RT at the current dose, TGF-ß3 treatment increased acute H&N radiation toxicities and increased mortality. Furthermore, TGF-ß3 treatment may increase the severity of radiation-induced salivary gland fibrosis.

Isoform-selective TGF-ß3 inhibition for systemic sclerosis.

BACKGROUND: Transforming growth factor ß (TGF-ß) is implicated as a key mediator of pathological fibrosis, but its pleiotropic activity in a range of homeostatic functions presents challenges to its safe and effective therapeutic targeting. There are three isoforms of TGF-ß, TGF-ß1, TGF-ß2, and TGF-ß3, which bind to a common receptor complex composed of TGF-ßR1 and TGF-ßR2 to induce similar intracellular signals in vitro. We have recently shown that the cellular expression patterns and activation thresholds of TGF-ß2 and TGF-ß3 are distinct from those of TGF-ß1 and that selective short-term TGF-ß2 and TGF-ß3 inhibition can attenuate fibrosis in vivo without promoting excessive inflammation. Isoform-selective inhibition of TGF-ß may therefore provide a therapeutic opportunity for patients with chronic fibrotic disorders. METHODS: Transcriptomic profiling of skin biopsies from patients with systemic sclerosis (SSc) from multiple clinical trials was performed to evaluate the role of TGF-ß3 in this disease. Antibody humanization, biochemical characterization, crystallization, and pre-clinical experiments were performed to further characterize an anti-TGF-ß3 antibody. FINDINGS: In the skin of patients with SSc, TGF-ß3 expression is uniquely correlated with biomarkers of TGF-ß signaling and disease severity. Crystallographic studies establish a structural basis for selective TGF-ß3 inhibition with a potent and selective monoclonal antibody that attenuates fibrosis effectively in vivo at clinically translatable exposures. Toxicology studies suggest that, as opposed to pan-TGF-ß inhibitors, this anti-TGF-ß3 antibody has a favorable safety profile for chronic administration. CONCLUSION: We establish a rationale for targeting TGF-ß3 in SSc with a favorable therapeutic index. FUNDING: This study was funded by Genentech, Inc.

Experimental study on the effect and mechanism of adipose stem cell-derived exosomes combined with botulinum toxin A on skin trauma in rats.

BACKGROUND: Adipose stem cell-derived exosomes (ADSC-EXO) and botulinum toxin type A (BTX-A) individually showed a therapeutic effect on skin wound repair. AIMS: This study investigated their synergistic effect on promoting skin wound healing in vitro and in vivo and the underlying molecular events. METHODS: ADSCs were isolated from Sprague-Dawley (SD) rats to obtain ADSC-EXO by ultrafiltration and ultracentrifugation and were confirmed using nanoparticle tracking analysis and transmission electron microscopy. Human skin fibroblasts (HSF) were cultured and treated with or without ADSC-EXO, BTX-A, or their combination. Changes in cell phenotypes and protein expression were analyzed using different in vitro assays, and a rat skin wound model was used to assess their in vivo effects. RESULTS: The isolated ADSC-EXO from primarily cultured ADSCs had a circular vesicle shape with a 30-180 nm diameter. Treatment of HSF with ADSC-EXO and/or BTX-A significantly accelerated HSF migration in vitro and skin wound healing in a rat model. Moreover, ADSC-EXO plus BTX-A treatment dramatically induced VEGFA expression but reduced COL III and COL I levels in vivo. ADSC-EXO and/or BTX-A treatment significantly upregulated TGF-ß3 expression on Day 16 after surgery but downregulated TGF-ß1 expression, suggesting that ADSC-EXO plus BTX-A promoted skin wound healing and reduced inflammatory cell infiltration. CONCLUSIONS: The ADSC-EXO plus BTX-A treatment demonstrated a synergistic effect on skin wound healing through upregulation of VEGF expression and the TGF-ß3/TGF-ß1 and COL III/COL I ratio.

GMFB/AKT/TGF-ß3 in Müller cells mediated early retinal degeneration in a streptozotocin-induced rat diabetes model.

Retinal degeneration, characterized by Müller cell gliosis and photoreceptor apoptosis, is considered an early event in diabetic retinopathy (DR). Our previous study proposed that GMFB may mediate diabetic retinal degeneration. This study identified GMFB as a sensitive and functional gliosis marker for DR. Compared to the wild type (WT) group, Gmfb knockout (KO) significantly improved visual function, attenuated gliosis, reduced the apoptosis of neurons, and decreased the mRNA levels of tumor necrosis factor α (Tnf-α) and interleukin-1ß (Il-1ß) in diabetic retinas. Tgf-ß3 was enriched by hub genes using RNA sequencing in primary WT and KO Müller cells. Gmfb KO significantly upregulated the transforming growth factor (TGF)-ß3 protein level via the AKT pathway. The protective effect of TGF-ß3 in the vitreous resulted in significantly improved visual function and decreased the number of apoptotic cells in the diabetic retina. The protection of Gmfb KO in primary Müller cells against high glucose (HG)-induced photoreceptor apoptosis was partially counteracted by TGF-ß3 antibody and administration of TGFBR1/2 inhibitors. Nuclear receptor subfamily 3 group C member 1 (NR3C1) binds to the promoter region of Gmfb and regulates Gmfb mRNA at the transcriptional level. NR3C1 was increased in the retinas of early diabetic rats but decreased in the retinas of late diabetic rats. N'-[(1E)-(3-Methoxyphenyl)Methylene]-3-Methyl-1H-Pyrazole-5-Carbohydrazide (DS-5) was identified as an inhibitor of GMFB, having a protective role in DR. We demonstrated that GMFB/AKT/TGF-ß3 mediated early diabetic retinal degeneration in diabetic rats. This study provides a novel therapeutic strategy for treating retinal degeneration in patients with DR.

TGF-ß1 and TGF-ß3, but not TGF-ß2, are upregulated in the ovaries of ovarian hyperstimulation syndrome†.

Ovarian hyperstimulation syndrome (OHSS) is a life-threatening and potentially fatal complication during in vitro fertilization treatment. The levels of transforming growth factor-ß1 (TGF-ß1) are upregulated in human follicular fluid and granulosa-lutein cells (hGL) of OHSS patients and could contribute to the development of OHSS by downregulating steroidogenic acute regulatory protein (StAR) expression. However, whether the same is true for the other two members of the TGF-ß family, TGF-ß2 and -ß3, remains unknown. We showed that all three TGF-ß isoforms were expressed in human follicular fluid. In comparison, TGF-ß1 was expressed at the highest level, followed by TGF-ß2 and TGF-ß3. Compared to non-OHSS patients, follicular fluid levels of TGF-ß1 and TGF-ß3 were significantly upregulated in OHSS patients. The same results were observed in mRNA levels of TGF-ß isoforms in hGL cells and ovaries of OHSS rats. In addition, StAR mRNA levels were upregulated in hGL cells of OHSS patients and the ovaries of OHSS rats. Treatment cells with TGF-ß isoforms downregulated the StAR expression with a comparable effect. Moreover, activations of SMAD3 signaling were required for TGF-ß isoforms-induced downregulation of StAR expression. This study indicates that follicular fluid TGF-ß1 and TGF-ß3 levels could be used as biomarkers and therapeutic targets for the OHSS.

Mechanistic study on ursolic acid inhibiting the growth of colorectal cancer cells through the downregulation of TGF-ß3 by miR-140-5p.

Colorectal cancer (CRC) is a common digestive tract tumor with a high incidence and a poor prognosis. Traditional chemotherapy drugs are usually accompanied by unpleasant side effects, highlighting the importance of exploring new adjunctive drugs. In this study, we aimed to explore the role of ursolic acid (UA) in CRC cells. Specifically, HT-29 cells were treated with UA at different concentrations (10, 20, 30, and 40 µM), and the expression of miR-140-5p, tumor growth factor-ß3 (TGF-ß3), ß-catenin, and cyclin D1 was determined by real-time quantitative PCR. The cell cycle and apoptosis were checked by flow cytometry, and cell proliferation was detected by Cell Counting Kit-8 assay. The HT-29 cell model was established through overexpression (miR-140-5p mimics) and interference (miR-140-5p inhibitor) of miR-140-5p. Western blot was used to detect the protein expression of TGF-ß3. We found that UA could inhibit the proliferation of HT-29 cells, block cells in the G1 phase, and promote cell apoptosis. After UA treatment, the expression of miR-140-5p increased and TGF-ß3 decreased. Notably, miR-140-5p downregulated the expression of TGF-ß3, while the overexpression of miR-140-5p exerted a similar function to UA in HT-29 cells. Additionally, the messenger RNA expression of TGF-ß3, ß-catenin, and cyclin D1 was decreased in HT-29 cells after UA treatment. In conclusion, UA inhibited CRC cell proliferation and cell cycle and promoted apoptosis by regulating the miR-140-5p/TGF-ß3 axis, which may be related to the inhibition of Wnt/ß-catenin signaling pathway.

Fabrication of blended nanofibrous cardiac patch transplanted with TGF-ß3 and human umbilical cord MSCs-derived exosomes for potential cardiac regeneration after acute myocardial infarction.

Acute myocardial infarction (AMI) is a common cardiovascular condition that progressively results in heart failure. In the present study, we have designed to load transforming growth factor beta 3 (TGF-ß3) and cardio potential exosomes into the blended polycaprolactone/type I collagen (PCL/COL-1) nanofibrous patch (Exo@TGF-ß3@NFs) and examined its feasibility for cardiac repair. The bioactivity of the developed NFs towards the migration and proliferation of human umbilical vein endothelial cells was determined using in vitro cell compatibility assays. Additionally, Exo@TGF-ß3/NFs showed up-regulation of genes involved in angiogenesis and mesenchymal differentiations in vitro. The in vivo experiments performed 4 weeks after transplantation showed that the Exo@TGF-ß3@NFs had a higher LV ejection fraction and fraction shortening functions. Subsequently, it has been determined that Exo@TGF-ß3@NFs significantly reduced AMI size and fibrosis and increased scar thickness. The developed NFs approach will become a useful therapeutic approach for the treatment of AMI.

Heterogeneous response to TGF-ß1/3 isoforms in fibroblasts of different origins: implications for wound healing and tumorigenesis.

Identification of therapeutic targets for treating fibrotic diseases and cancer remains challenging. Our study aimed to investigate the effects of TGF-ß1 and TGF-ß3 on myofibroblast differentiation and extracellular matrix deposition in different types of fibroblasts, including normal/dermal, cancer-associated, and scar-derived fibroblasts. When comparing the phenotype and signaling pathways activation we observed extreme heterogeneity of studied markers across different fibroblast populations, even within those isolated from the same tissue. Specifically, the presence of myofibroblast and deposition of extracellular matrix were dependent on the origin of the fibroblasts and the type of treatment they received (TGF-ß1 vs. TGF-ß3). In parallel, we detected activation of canonical signaling (pSMAD2/3) across all studied fibroblasts, albeit to various extents. Treatment with TGF-ß1 and TGF-ß3 resulted in the activation of canonical and several non-canonical pathways, including AKT, ERK, and ROCK. Among studied cells, cancer-associated fibroblasts displayed the most heterogenic response to TGF-ß1/3 treatments. In general, TGF-ß1 demonstrated a more potent activation of signaling pathways compared to TGF-ß3, whereas TGF-ß3 exhibited rather an inhibitory effect in keloid- and hypertrophic scar-derived fibroblasts suggesting its clinical potential for scar treatment. In summary, our study has implications for comprehending the role of TGF-ß signaling in fibroblast biology, fibrotic diseases, and cancer. Future research should focus on unraveling the mechanisms beyond differential fibroblast responses to TGF-ß isomers considering inherent fibroblast heterogeneity.

The Role of TGF-ß3 in Radiation Response.

Transforming growth factor-beta 3 (TGF-ß3) is a ubiquitously expressed multifunctional cytokine involved in a range of physiological and pathological conditions, including embryogenesis, cell cycle regulation, immunoregulation, and fibrogenesis. The cytotoxic effects of ionizing radiation are employed in cancer radiotherapy, but its actions also influence cellular signaling pathways, including that of TGF-ß3. Furthermore, the cell cycle regulating and anti-fibrotic effects of TGF-ß3 have identified it as a potential mitigator of radiation- and chemotherapy-induced toxicity in healthy tissue. This review discusses the radiobiology of TGF-ß3, its induction in tissue by ionizing radiation, and its potential radioprotective and anti-fibrotic effects.

Up-regulation of CREB-1 regulates tendon adhesion in the injury tendon healing through the CREB-1/TGF-ß3 signaling pathway.

AIM: To explore the mechanism of the healing of tendon tissue and anti-adhesion, and to discuss the role of the transforming growth factor-ß3 (TGF-ß3)/cAMP response element binding protein-1 (CREB-1) signaling pathway in the healing process of tendons. METHOD: All mice were divided into four groups of 1, 2, 4, and 8 weeks respectively. Each time group was divided into four treatment groups: the amplification group, the inhibition group, the negative group, and the control group. When the tendon injury model was established, the CREB-1 virus was injected into the tendon injury parts. A series of methods such as gait behaviourism, anatomy, histological examination, immunohistochemical examination and collagen staining were employed to assess the tendon healing and the protein expression of TGF-ß3, CREB-1, Smad3/7 and type I/III collagen (COL-I/III). CREB-1 virus was sent to tendon stem cells to assess the protein expression of TGF-ß1, TGF-ß3, CREB-1, COL-I/III by methods such as immunohistochemistry and Western blot. RESULTS: The amplification group showed better gait behaviourism than the inhibition group in the healing process. The amplification group also had less adhesion than the negative group. Hematoxylin-eosin (HE) staining of tendon tissue sections showed that the number of fibroblasts in the amplification group was less than the inhibition group, and the immunohistochemical results indicated that the expression of TGF-ß3, CREB-1, and Smad7 at each time point was higher than the inhibition group. The expression of COL-I/III and Smad3 in the amplification group was lower than the inhibition group at all time points. The collagen staining indicated that the ratio of type I/III collagen in the amplification group was higher than the negative group at 2,4,8 week. The CREB-1 amplification virus could promote the protein expression of TGF-ß3, CREB-1 and inhibit the protein expression of TGF-ß1 and COL-I/III in the tendon stem cells. CONCLUSION: In the process of tendon injury healing, CREB-1 could promote the secretion of TGF-ß3, so as to promote the tendon healing and have the effect of anti-adhesion in tendons. It might provide new intervention targets for anti-adhesion treatment of tendon injuries.

Extracellular Vesicles Derived from Auricular Chondrocytes Facilitate Cartilage Differentiation of Adipose-Derived Mesenchymal Stem Cells.

PURPOSE: Adipose-derived mesenchymal stem cell (ADSC)-based therapies have been utilized for cartilage regeneration because of their multi-lineage differentiation ability. However, commonly used cartilage inducers such as the transforming growth factor beta-3 (TGF-ß3) may be prone to cartilage dedifferentiation and hypertrophy. The directional differentiation of elastic cartilage is limited nowadays. Extracellular vesicles (EVs) have been reported to influence the specific differentiation of mesenchymal stem cells (MSCs) by reflecting the composition of the parental cells. However, the role of auricular chondrogenic-derived EVs (AC-EVs) in elastic chondrogenic differentiation of ADSCs has not yet been reported. RESULTS: AC-EVs isolated from the external ears of swine exhibited a positive effect on cell proliferation and migration. Furthermore, AC-EVs efficiently promoted chondrogenic differentiation of ADSCs in pellet culture, as shown by the elevated levels of COL2A1, ACAN, and SOX-9 expression. Moreover, there was a significantly higher expression of elastin and a lower expression of the fibrotic marker COL1A1 in comparison with that achieved with TGF-ß3. The staining results demonstrated that AC-EVs promoted the deposition of cartilage-specific matrix, which is in good concordance with the real-time polymerase chain reaction (RT-PCR) results. CONCLUSIONS: Auricular chondrogenic-derived EVs are a crucial component in elastic chondrogenic differentiation and other biological behaviors of ADSCs, which may be a useful ingredient for cartilage tissue engineering and external ear reconstruction. NO LEVEL ASSIGNED: This journal requires that authors 42 assign a level of evidence to each submission to which 43 Evidence-Based Medicine rankings are applicable. This 44 excludes Review Articles, Book Reviews, and manuscripts 45 that concern Basic Science, Animal Studies, Cadaver 46 Studies, and Experimental Studies. For a full description of 47 these Evidence-Based Medicine ratings, please refer to the 48 Table oôf Contents or the online Instructions to Authors 49 www.springer.com/00266 .

Adipocyte and Adipokines Promote a Uterine Leiomyoma Friendly Microenvironment.

Uterine leiomyomas are the most common benign tumors of the female reproductive system. Obese individuals have a higher burden of uterine leiomyoma, yet the mechanism relating obesity and leiomyoma development remains unknown. In this study, we observe the effect of adipocyte coculture and leptin treatment on human myometrium and leiomyoma cells. We isolated primary leiomyoma and myometrium cells from hysterectomy or myomectomy patients. Protein expression levels of phosphorylated ERK1/2/total ERK1/2, phosphorylated STAT3/total STAT3, and phosphorylated AKT1/2/3/total AKT1/2/3 were quantified using immunoblotting in immortalized and primary leiomyoma and myometrial cells cocultured with human adipocytes and treated with leptin. An enzyme-linked immunosorbent assay (ELISA) was used to assess pro-inflammatory, fibrotic, and angiogenic factors in immortalized human myometrium and leiomyoma cells treated with leptin. The effects of STAT3, ERK, and AKT inhibitors were assessed in leiomyoma cell lines additionally cultured with adipocytes. Adipocyte coculture and leptin treatment increases the expression of JAK2/STAT3, MAPK/ERK, and PI3K/AKT signaling while inhibitors suppressed this effect. Leptin induces a tumor-friendly microenvironment through upregulation of pro-inflammatory (IFNγ, IL-8, IL-6, GM-CSF, MCP-1, and TNF-α), fibrotic (TGF-ß1, TGF-ß2, and TGF-ß3), and angiogenic (VEGF-A, HGF, and Follistatin) factors in human leiomyoma cells. Furthermore, adipocyte coculture and leptin treatment increases leiomyoma cells growth through activation of MAPK/ERK, JAK2/STAT3, and PI3k/AKT signaling pathways. Finally, STAT3, ERK, and AKT inhibitor treatment suppressed PCNA, TNF-α, TGF-ß3, and VEGF-A intracellular staining intensity in both adipocyte coculture and leptin treated leiomyoma cells. These findings suggest that, in obese women, adipocyte secreted hormone or adipocytes may contribute to leiomyoma development and growth by activating leptin receptor signaling pathways.

TGF-ß-3 Induces Different Effects from TGF-ß-1 and -2 on Cellular Metabolism and the Spatial Properties of the Human Trabecular Meshwork Cells.

To compare the effects among three TGF-ß isoforms (TGF-ß-1, TGF-ß-2, and TGF-ß-3) on the human trabecular meshwork (HTM), two-dimensional (2D) and three-dimensional (3D) cultures of commercially available certified immortalized HTM cells were used, and the following analyses were conducted: (1) trans-endothelial electrical resistance (TEER) and FITC dextran permeability measurements (2D); (2) a real-time cellular metabolic analysis (2D); (3) analysis of the physical property of the 3D HTM spheroids; and (4) an assessment of the gene expression levels of extracellular matrix (ECM) components (2D and 3D). All three TGF-ß isoforms induced a significant increase in TEER values and a relative decrease in FITC dextran permeability in the 2D-cultured HTM cells, but these effects were the most potent in the case of TGF-ß-3. The findings indicated that solutions containing 10 ng/mL of TGF-ß-1, 5 ng/mL of TGF-ß-2, and 1 ng/mL of TGF-ß-3 had nearly comparable effects on TEER measurements. However, a real-time cellular metabolic analysis of the 2D-cultured HTM cells under these concentrations revealed that TGF-3-ß induced quite different effects on the metabolic phenotype, with a decreased ATP-linked respiration, increased proton leakage, and decreased glycolytic capacity compared with TGF-ß-1 and TGF-ß-2. In addition, the concentrations of the three TGF-ß isoforms also caused diverse effects on the physical properties of 3D HTM spheroids and the mRNA expression of ECMs and their modulators, in many of which, the effects of TGF-ß-3 were markedly different from TGF-ß-1 and TGF-ß-2. The findings presented herein suggest that these diverse efficacies among the TGF-ß isoforms, especially the unique action of TGF-ß-3 toward HTM, may induce different effects within the pathogenesis of glaucoma.