TGF-ß/Smad signaling pathway in fatty liver disease: a case-control study.

BACKGROUND: Fatty liver disease is a metabolic disorder that recently has been classified into two categories: metabolic dysfunction-associated fatty liver disease (MAFLD) and non-MAFLD. TGF-ß signaling pathway is likely a significant factor in the pathogenesis of this condition, exerting its effects through its downstream signaling proteins, Smad2/3. Accordingly, this study aimed to investigate the TGF-ß signaling pathway in the white blood cells (WBCs) of patients with MAFLD compared to those with non-MAFLD and control groups. METHODS AND RESULTS: In this study, 41 patients with fatty liver were evaluated, comprising 22 patients with MAFLD and 19 patients with non-MAFLD, and compared to 22 healthy controls. Gene expression of TGF-ß1, TGF-ß3, and CTGF were quantified using qRT-PCR, and the protein expressions of Smad2/3 and P-Smad2/3 were analyzed using western blotting. Gene expression analysis revealed a significant decrease in the gene expressions of the TGF-ß1 and TGF-ß3 and an increase in CTGF gene expression in patients with MAFLD and non-MAFLD compared to the control group. Notably, the Smad2/3 protein expression was significantly higher in the non-MAFLD group compared to the control group (P < 0.05). On the other hand, the P-smad2/3 protein expression was significantly elevated in the MAFLD group compared to the control group (P < 0.001). CONCLUSIONS: TGF-ß signaling pathway in WBCs of patients with fatty liver are affected by a complex signaling pathway. However, metabolic factors most probably affect TGF-ß1 gene expression and its downstream signaling proteins more than TGF-ß3.

Protective role of transforming growth factor-Β3 (TGF-Β3) in the formation of radiation-induced capsular contracture around a breast implant: In vivo experimental study.

Postmastectomy radiotherapy causes capsular contracture due to fibroproliferation of the capsular tissue around the implant. In fibrosis, unlike normal wound healing, structural and functional disorders are observed in the tissues caused by excessive/irregular accumulation of extracellular matrix proteins. It has been reported that transforming growth factor-ß3 (TGF-ß3) prevents and reverses fibrosis in various tissues or provides scarless healing with its antifibrotic effect. Additionally, TGF-ß3 has been shown to reduce fibrosis in radiotherapy-induced fibrosis syndrome. However, no study in the literature investigates the effects of exogenously applied TGF-ß3 on capsular contracture in aesthetic or reconstructive breast implant application. TGF-ß3, which has a very short half-life, has low bioavailability with parenteral administration. Within the scope of this study, free TGF-ß3 was loaded into the nanoparticles to increase its low bioavailability and extend its duration of action by providing controlled release. The aim of this study is to investigate the preventive/improving effects of radiation induced capsular contracture using chitosan film formulations containing TGF-ß3 loaded poly(lactic-co-glycolic acid)-b-poly(ethylene glycol) (PLGA-b-PEG) nanoparticles in implant-based breast reconstruction. In the characterization studies of nanoparticles, the particle size and zeta potential of the TGF-ß3-loaded PLGA-b-PEG nanoparticle formulation selected to be used in the treatment group were found to be 123.60 ± 2.09 nm and -34.87 ± 1.42 mV, respectively. The encapsulation efficiency of the formulation was calculated as 99.91 %. A controlled release profile was obtained in in vitro release studies. Chitosan film formulations containing free TGF-ß3 or TGF-ß3-loaded PLGA-b-PEG nanoparticles were used in in vivo studies. In animal studies, rats were randomly distributed into 6 groups (n = 8) as sham, implant, implant + radiotherapy, implant + radiotherapy + chitosan film containing unloaded nanoparticles, implant + radiotherapy + chitosan film containing free TGF-ß3, implant + radiotherapy + chitosan film containing TGF-ß3 loaded nanoparticle. In all study groups, a 2 cm incision was made along the posterior axillary line at the thoracic vertebral level in rats to reach the lateral edge of the latissimus dorsi. The fascial attachment to the chest wall was then bluntly dissected to create a pocket for the implants. In the treatment groups, the wound was closed after films were placed on the outer surface of the implants. After administering prophylactic antibiotics, rats were subjected to irradiation with 10 Gy photon beams targeted to each implant site. Each implant and the surrounding excised tissue were subjected to the necessary procedures for histological (capsule thickness, cell density), immunohistochemical, and biochemical (α-SMA, vimentin, collagen type I and type III, TGF-ß1 and TGF-ß3: expression level/protein level) examinations. It was determined that the levels of TGF-ß1 and TGF-ß3 collagen type III, which decreased as a result of radiotherapy, were brought to the control level with free TGF-ß3 film and TGF-ß3 nanoparticle film formulations. Histological analyses, consistent with biochemical analyses, showed that thick collagen and fibrosis, which increased with radiotherapy, were brought to the control level with free TGF-ß3 film and TGF-ß3 nanoparticle film treatments. In biochemical analyses, the decrease in thick collagen was compatible with the decrease in the collagen type I/type III ratio in the free TGF-ß3 film and TGF-ß3 nanoparticle film groups. Changes in protein expression show that TGF-ß3 loaded nanoparticles are more successful than free TGF-ß3 in wound healing. In line with these results and the literature, it is thought that the balance of TGF-ß1 and TGF-ß3 should be maintained to ensure scarless wound healing with no capsule contracture.

Aptamer-Modified Tetrahedral Framework Nucleic Acid Synergized with TGF-ß3 to Promote Cartilage Protection in Osteoarthritis by Enhancing Chondrogenic Differentiation of MSCs.

Characterized by progressive and irreversible degeneration of the articular cartilage (AC), osteoarthritis (OA) is the most common chronic joint disease, and there is no cure for OA at present. Recent studies suggest that enhancing the recruitment of endogenous mesenchymal stem cells (MSCs) to damaged cartilage is a promising therapeutic strategy for cartilage repair. Tetrahedral framework nucleic acid (tFNA) is a novel DNA nanomaterial and has shown great potential in the field of biomedical science. Transforming growth factor-beta 3 (TGF-ß3), a vital member of the highly conserved TGF-ß superfamily, is considered to induce chondrogenesis. A 66-base DNA aptamer named HM69 is reported to identify and recruit MSCs. In this study, aptamer HM69-modified tFNAs were successfully self-assembled and used to load TGF-ß3 when the disulfide bonds combined. We confirmed the successful synthesis of the final composition, HM69-tFNA@TGF-ß3 (HTT), by PAGE, dynamic light scattering, and atomic force microscopy. The results of in vitro experiments showed that HTT effectively induced MSC proliferation, migration, and chondrogenic differentiation. In addition, HTT-treated MSCs were shown to protect the OA chondrocytes. In DMM mice, the injection of HTT improved the therapeutic outcome of mouse pain symptoms and AC degeneration. In conclusion, this study innovatively used the disulfide bonds combined with TGF-ß3 and tFNA, and an additional sequence HM69 was loaded on tFNA for the better-targeted recruitment of MSCs. HTT demonstrated its role in promoting the chondrogenesis of MSCs and cartilage protection, indicating that it might be promising for OA therapy.

Adapting to the acidic environment of the NP: RADA16-PLGA (TGF-ß3) induces chondrogenic differentiation of BMSCs.

Aim: RADA16-PLGA composite scaffolds constructed with simultaneous loading of BMSCs and TGF-ß3 and explored their ability for chondrogenic differentiation in vitro.Methods: The performance of the composite scaffolds is assessed by rheometer assay, electron microscopic structural observation and ELISA release assay. The biosafety of the composite scaffolds is assessed by cytocompatibility assay and cell migration ability. The chondrogenic differentiation ability of composite scaffolds is evaluated by Alisin blue staining, PCR and immunofluorescence staining.Results: The composite scaffold has a good ECM-like structure, the ability to control the release of TGF-ß3 and good biocompatibility. More importantly, the composite scaffolds can induce the differentiation of BMSCs to chondrocytes.Conclusion: Composite scaffolds are expected to enhance the endogenous NP repair process.

[Box: see text].

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.

Enhanced effects of slowly co-released TGF-ß3 and BMP-2 from biomimetic calcium phosphate-coated silk fibroin scaffolds in the repair of osteochondral defects.

Bioactive agents have demonstrated regenerative potential for cell-free bone tissue engineering. Nevertheless, certain challenges persist, including ineffective delivery methods and confined therapeutic potency. Here, we demonstrated that the biomimetic calcium phosphate coating system (BioCaP) could effectively uptake and slowly release the incorporated bioactive agents compared to the surface absorption system via osteoclast-mediated degradation of BioCaP coatings. The release kinetics were determined as a function of time. The release rate was stable without remarkable burst release during the first 1 day, followed by a sustained release from day 7 to day 19. Then, we developed the bi-functional BioCaP-coated silk fibroin scaffolds enabling the effective co-delivery of TGF-ß3 and BMP-2 (SFI-T/SFI-B) and the corresponding slow release of TGF-ß3 and BMP-2 exhibited superior potential in promoting chondrogenesis and osteogenesis without impairing cell vitality in vitro. The SFI-T/SFI-B scaffolds could improve cartilage and bone regeneration in 5 × 4 mm rabbit osteochondral (OC) defect. These findings indicate that the biomimetic calcium-phosphate coated silk fibroin scaffolds with slowly co-released TGF-ß3 and BMP-2 effectively promote the repair of OC defects, hence facilitating the future clinical translation of controlled drug delivery in tissue engineering.

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.

Delivery of TGFß3 from Magnetically Responsive Coaxial Fibers Reduces Spinal Cord Astrocyte Reactivity In Vitro.

A spinal cord injury (SCI) compresses the spinal cord, killing neurons and glia at the injury site and resulting in prolonged inflammation and scarring that prevents regeneration. Astrocytes, the main glia in the spinal cord, become reactive following SCI and contribute to adverse outcomes. The anti-inflammatory cytokine transforming growth factor beta 3 (TGFß3) has been shown to mitigate astrocyte reactivity; however, the effects of prolonged TGFß3 exposure on reactive astrocyte phenotype have not yet been explored. This study investigates whether magnetic core-shell electrospun fibers can be used to alter the release rate of TGFß3 using externally applied magnetic fields, with the eventual application of tailored drug delivery based on SCI severity. Magnetic core-shell fibers are fabricated by incorporating superparamagnetic iron oxide nanoparticles (SPIONs) into the shell and TGFß3 into the core solution for coaxial electrospinning. Magnetic field stimulation increased the release rate of TGFß3 from the fibers by 25% over 7 days and released TGFß3 reduced gene expression of key astrocyte reactivity markers by at least twofold. This is the first study to magnetically deliver bioactive proteins from magnetic fibers and to assess the effect of sustained release of TGFß3 on reactive astrocyte phenotype.

The Presence of TGFß3 in Human Ovarian Intrafollicular Fluid and Its Involvement in Thromboxane Generation in Follicular Granulosa Cells through a Canonical TGFßRI, Smad2/3 Signaling Pathway and COX-2 Induction.

Ovarian follicular fluid (FF) has a direct impact on oocyte quality, playing key roles in fertilization, implantation, and early embryo development. In our recent study, we found FF thromboxane (TX) to be a novel factor inversely correlated with oocyte maturation and identified thrombin, transforming growth factor ß (TGFß), TNF-α, and follicular granulosa cells (GCs) as possible contributors to FF TX production. Therefore, this study sought to investigate the role of TGFß3 in regulating TX generation in human ovarian follicular GCs. TGFß3 was differentially and significantly present in the FF of large and small follicles obtained from IVF patients with average concentrations of 68.58 ± 12.38 and 112.55 ± 14.82 pg/mL, respectively, and its levels were correlated with oocyte maturity. In an in vitro study, TGFß3 induced TX generation/secretion and the converting enzyme-COX-2 protein/mRNA expression both in human HO23 and primary cultured ovarian follicular GCs. While TGFßRI and Smad2/3 signaling was mainly required for COX-2 induction, ERK1/2 appeared to regulate TX secretion. The participation of Smad2/3 and COX-2 in TGFß3-induced TX generation/secretion could be further supported by the observations that Smad2/3 phosphorylation and nuclear translocation and siRNA knockdown of COX-2 expression compromised TX secretion in GCs challenged with TGFß3. Taken together, the results presented here first demonstrated that FF TGFß3 levels differ significantly in IVF patients' large preovulatory and small mid-antral follicles and are positively associated with oocyte maturation. TGFß3 can provoke TX generation by induction of COX-2 mRNA/protein via a TGFßR-related canonical Smad2/3 signaling pathway, and TX secretion possibly by ERK1/2. These imply that TGFß3 is one of the inducers for yielding FF TX in vivo, which may play a role in folliculogenesis and oocyte maturation.

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.

Endocardial HDAC3 is required for myocardial trabeculation.

Failure of proper ventricular trabeculation is often associated with congenital heart disease. Support from endocardial cells, including the secretion of extracellular matrix and growth factors is critical for trabeculation. However, it is poorly understood how the secretion of extracellular matrix and growth factors is initiated and regulated by endocardial cells. We find that genetic knockout of histone deacetylase 3 in the endocardium in mice results in early embryo lethality and ventricular hypotrabeculation. Single cell RNA sequencing identifies significant downregulation of extracellular matrix components in histone deacetylase 3 knockout endocardial cells. Secretome from cultured histone deacetylase 3 knockout mouse cardiac endothelial cells lacks transforming growth factor ß3 and shows significantly reduced capacity in stimulating cultured cardiomyocyte proliferation, which is remarkably rescued by transforming growth factor ß3 supplementation. Mechanistically, we identify that histone deacetylase 3 knockout induces transforming growth factor ß3 expression through repressing microRNA-129-5p. Our findings provide insights into the pathogenesis of congenital heart disease and conceptual strategies to promote myocardial regeneration.

FasL impacts Tgfb signaling in osteoblastic cells.

Fas ligand (FasL, CD178) belongs to classical apoptotic molecules, however, recent evidence expands the spectrum of FasL functions into non-apoptotic processes which also applies for the bone. Tgfb subfamily members (Tgfb1, Tgfb2, Tgfb3) represent major components in osteogenic pathways and extracellular matrix. Their possible association with FasL has not yet been investigated but can be postulated. To test such a hypothesis, FasL deficient (gld) calvaria-derived cells were examined with a focus on the expression of Tgfb receptor ligands. The qPCR analysis revealed significantly increased expression of Tgfb1, Tgfb2 and Tgfb3 in gld cells. To check the vice versa effect, the gld cells were stimulated by soluble FasL. As a consequence, a dramatic decrease in expression levels of all three ligands was observed. This phenomenon was also confirmed in IDG-SW3 (osteoblastic cells of endochondral origin). TFLink gateway identified Fosl2 as an exclusive candidate of FasL capable to impact expression of all three Tgfb ligands. However, Fosl2 siRNA did not cause any significant changes in expression of Tgfb ligands. Therefore, the upregulation of the three ligands is likely to occur separately. In this respect, we tested the only exclusive candidate transcription factor for Tgfb3, Prrx1. Additionally, an overlapping candidate for Tgfb1 and Tgfb2, Mef2c capable to modulate expression of sclerostin, was examined. Prrx1 as well as Mef2c were found upregulated in gld samples and their expression decreased after addition of FasL. The same effect of FasL treatment was observed in the IDG-SW3 model. Taken together, FasL deficiency causes an increase in the expression of Tgfb ligands and stimulation by FasL reduces Tgfb expression in osteoblastic cells. The candidates mediating the effect comprise Prrx1 for Tgfb3 and Mef2c for Tgfb1/2. These results indicate FasL as a novel cytokine interfering with Tgfb signaling and thus the complex osteogenic network. The emerging non-apoptotic functions of FasL in bone development and maintenance should also be considered in treatment strategies such as the anti-osteoporotic factor.

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.

Differential expression of small bowel TGFß1 and TGFß3 characterizes intestinal strictures in patients with fibrostenotic Crohn's disease.

Small bowel strictures remain a debilitating consequence of Crohn's disease and contribute to poor outcomes for patients. Recently, TGFß has been identified as an important driver of intestinal fibrosis. We studied the localization of TGFß isoforms in ileal strictures of patients with Crohn's disease using in situ hybridization to understand TGFß's role in stricture formation. The mucosa of strictures was characterized by higher TGFß1 while the stricture submucosa showed higher TGFß3 compared to normal ileum from patients without Crohn's disease (p = 0.02 and p = 0.044, respectively). We correlated these findings with single-cell transcriptomics which demonstrated that TGFß3 transcripts overall are very rare, which may partially explain why its role in intestinal fibrosis has remained unclear to date. There were no significant differences in fibroblast or B cell TGFß1 and/or TGFß3 expression in inflamed vs. noninflamed ileum. We discuss the implications of these findings for therapeutic development strategies to treat patients with fibrostenotic Crohn's disease.

Spatiotemporal Evolution of Developing Palate in Mice.

The intricate formation of the palate involves a series of complex events, yet its mechanistic basis remains uncertain. To explore major cell populations in the palate and their roles during development, we constructed a spatiotemporal transcription landscape of palatal cells. Palate samples from C57BL/6 J mice at embryonic days 12.5 (E12.5), 14.5 (E14.5), and 16.5 (E16.5) underwent single-cell RNA sequencing (scRNA-seq) to identify distinct cell subsets. In addition, spatial enhanced resolution omics-sequencing (stereo-seq) was used to characterize the spatial distribution of these subsets. Integrating scRNA-seq and stereo-seq with CellTrek annotated mesenchymal and epithelial cellular components of the palate during development. Furthermore, cellular communication networks between these cell subpopulations were analyzed to discover intercellular signaling during palate development. From the analysis of the middle palate, both mesenchymal and epithelial populations were spatially segregated into 3 domains. The middle palate mesenchymal subpopulations were associated with tooth formation, ossification, and tissue remodeling, with initial state cell populations located proximal to the dental lamina. The nasal epithelium of the palatal shelf exhibited richer humoral immune responses than the oral side. Specific enrichment of Tgfß3 and Pthlh signals in the midline epithelial seam at E14.5 suggested a role in epithelial-mesenchymal transition. In summary, this study provides high-resolution transcriptomic information, contributing to a deeper mechanistic understanding of palate biology and pathophysiology.

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.

Engineered Microenvironmental Cues from Fiber-Reinforced Hydrogel Composites Drive Tenogenesis and Aligned Collagen Deposition.

Effective tendon regeneration following injury is contingent on appropriate differentiation of recruited cells and deposition of mature, aligned, collagenous extracellular matrix that can withstand the extreme mechanical demands placed on the tissue. As such, myriad biomaterial approaches have been explored to provide biochemical and physical cues that encourage tenogenesis and template aligned matrix deposition in lieu of dysfunctional scar tissue formation. Fiber-reinforced hydrogels present an ideal biomaterial system toward this end given their transdermal injectability, tunable stiffness over a range amenable to tenogenic differentiation of progenitors, and capacity for modular inclusion of biochemical cues. Here, tunable and modular, fiber-reinforced, synthetic hydrogels are employed to elucidate salient microenvironmental determinants of tenogenesis and aligned collagen deposition by tendon progenitor cells. Transforming growth factor ß3 drives a cell fate switch toward pro-regenerative or pro-fibrotic phenotypes, which can be biased toward the former by culture in softer microenvironments or inhibition of the RhoA/ROCK activity. Furthermore, studies demonstrate that topographical anisotropy in fiber-reinforced hydrogels critically mediates the alignment of de novo collagen fibrils, reflecting native tendon architecture. These findings inform the design of cell-free, injectable, synthetic hydrogels for tendon tissue regeneration and, likely, that of a range of load-bearing connective tissues.

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.

Lack of Elevated Expression of TGFß3 Contributes to the Delay of Epithelial Wound Healing in Diabetic Corneas.

Purpose: To investigate the mechanisms underlying the differential roles of TGFß1 and TGFß3 in accelerating corneal epithelial wound healing (CEWH) in diabetic (DM) corneas, with normoglycemia (NL) corneas as the control. Methods: Two types of diabetic mice, human corneal organ cultures, mouse corneal epithelial progenitor cell lines, and bone marrow-derived macrophages (BMDMs) were employed to assess the effects of TGFß1 and TGFß3 on CEWH, utilizing quantitative PCR, western blotting, ELISA, and whole-mount confocal microscopy. Results: Epithelial debridement led to an increased expression of TGFß1 and TGFß3 in cultured human NL corneas, but only TGFß1 in DM corneas. TGFß1 and TGFß3 inhibition was significantly impeded, but exogenous TGFß1 and, more potently, TGFß3 promoted CEWH in cultured TKE2 cells and in NL and DM C57BL6 mouse corneas. Wounding induced similar levels of p-SMAD2/SMAD3 in NL and DM corneas but weaker ERK1/2, Akt, and EGFR phosphorylation in DM corneas compared to NL corneas. Whereas TGFß1 augmented SMAD2/SMAD3 phosphorylation, TGFß3 preferentially activated ERK, PI3K, and EGFR in healing DM corneas. Furthermore, TGFß1 and TGFß3 differentially regulated the expression of S100a9, PAI-1, uPA/tPA, and CCL3 in healing NL and DM corneas. Finally, TGFß1 induced the expression of M1 macrophage markers iNOS, CD86, and CTGF, whereas TGFß3 promoted the expression of M2 markers CD206 and NGF in BMDMs from db/db or db/+ mice. Conclusions: Hyperglycemia disrupts the balanced expression of TGFß3/TGFß1, resulting in delayed CEWH, including impaired sensory nerve regeneration in the cornea. Supplementing TGFß3 in DM wounds may hold therapeutic potential for accelerating delayed wound healing in diabetic patients.