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Background/Objective: We assessed the analgesic and anti-inflammatory effects of kale extract (500 mg anthocyanin equivalent) in patients after mandibular molar surgery. Methods: In our randomized clinical trial, postoperative subjects (n = 20) aged 18–25 years old took kale extract or ibuprofen (400 mg) capsules for 7 days, or vice versa, after surgical removal of each impacted tooth. Their pain intensity was then assessed using a visual analogue scale (VAS). Moreover, salivary α-amylase (AA) activity, matrix metalloproteinase 9 (MMP-9) and transforming growth factor beta2 (TGF-β2) concentrations were measured. Levels of VAS and AA decreased 7 days after the first and second molar extractions in the two treatment groups. Results: The kale extract was more effective than ibuprofen. MMP-9 and TGF-β2 levels were reduced on days 4 and 7 following the two extractions in the kale group, whereas they were reduced on days 4 and 7 following the first extraction in the ibuprofen group. There was a positive correlation between MMP-9 and TGF-β2. Thus, the consumption of the kale extract exerted analgesic and anti-inflammatory effects during the postoperative period in patients who had undergone molar extractions. In conclusion, anthocyanin-abundant kale extract is preferable when administered in a postoperative course and could reduce the need for a prescription of ibuprofen. [ABSTRACT FROM AUTHOR]

Transforming growth factor-β (TGFβ) is a key driver of fibrogenesis. Three TGFβ isoforms (TGFβ1, TGFβ2, and TGFβ3) in mammals have distinct functions in embryonic development; however, the postnatal pathological roles and activation mechanisms of TGFβ2 and TGFβ3 have not been well characterized. Here, we show that the latent forms of TGFβ2 and TGFβ3 can be activated by integrin-independent mechanisms and have lower activation thresholds compared to TGFβ1. Unlike TGFB1, TGFB2 and TGFB3 expression is increased in human lung and liver fibrotic tissues compared to healthy control tissues. Thus, TGFβ2 and TGFβ3 may play a pathological role in fibrosis. Inducible conditional knockout mice and anti-TGFβ isoform-selective antibodies demonstrated that TGFβ2 and TGFβ3 are independently involved in mouse fibrosis models in vivo, and selective TGFβ2 and TGFβ3 inhibition does not lead to the increased inflammation observed with pan-TGFβ isoform inhibition. A cocrystal structure of a TGFβ2-anti-TGFβ2/3 antibody complex reveals an allosteric isoform-selective inhibitory mechanism. Therefore, inhibiting TGFβ2 and/or TGFβ3 while sparing TGFβ1 may alleviate fibrosis without toxicity concerns associated with pan-TGFβ blockade.

Epidermal lineages and injury induced regeneration are controlled by transcriptional programs coordinating cellular signaling and epigenetic regulators, but the mechanism remains unclear. Previous studies showed that conditional deletion of the transcriptional coactivator Mediator 1 (Med1) changes epidermal lineages and accelerates wound re-epithelialization. Here, we studied a molecular mechanism by which Med1 facilitates these processes, in particular, by focusing on TGFβ signaling through genome wide transcriptome analysis. The expression of the TGF ligands (Tgfβ1/β2) and their downstream target genes is decreased in both normal and wounded Med1 null skin. Med1 silencing in cultured keratinocytes likewise reduces the expression of the ligands (TGFβ1/β2) and diminishes activity of TGFβ signaling as shown by decreased p-Smad2/3. Silencing Med1 increases keratinocyte proliferation and migration in vitro. Epigenetic studies using chromatin immuno-precipitation and next generation DNA sequencing reveals that Med1 regulates transcription of TGFβ components by forming large clusters of enhancers called super-enhancers at the regulatory regions of the TGFβ ligand and SMAD3 genes. These results demonstrate that Med1 is required for the maintenance of the TGFβ signaling pathway. Finally, we show that pharmacological inhibition of TGFβ signaling enhances epidermal lineages and accelerates wound re-epithelialization in skin similar to that seen in the Med1 null mice, providing new insights into epidermal regeneration.

Central corneal thickness (CCT) is a highly heritable trait associated with complex eye diseases such as keratoconus and glaucoma. We perform a genome-wide association meta-analysis of CCT and identify 19 novel regions. In addition to adding support for known connective tissue-related pathways, pathway analyses uncover previously unreported gene sets. Remarkably, >20% of the CCT-loci are near or within Mendelian disorder genes. These included FBN1, ADAMTS2 and TGFB2 which associate with connective tissue disorders (Marfan, Ehlers-Danlos and Loeys-Dietz syndromes), and the LUM-DCN-KERA gene complex involved in myopia, corneal dystrophies and cornea plana. Using index CCT-increasing variants, we find a significant inverse correlation in effect sizes between CCT and keratoconus (r = -0.62, P = 5.30 × 10-5) but not between CCT and primary open-angle glaucoma (r = -0.17, P = 0.2). Our findings provide evidence for shared genetic influences between CCT and keratoconus, and implicate candidate genes acting in collagen and extracellular matrix regulation.

The impact of EGFR-mutant NSCLC precision therapy is limited by acquired resistance despite initial excellent response. Classic studies of EGFR-mutant clinical resistance to precision therapy were based on tumor rebiopsies late during clinical tumor progression on therapy. Here, we characterized a novel non-mutational early adaptive drug-escape in EGFR-mutant lung tumor cells only days after therapy initiation, that is MET-independent. The drug-escape cell states were analyzed by integrated transcriptomic and metabolomics profiling uncovering a central role for autocrine TGFβ2 in mediating cellular plasticity through profound cellular adaptive Omics reprogramming, with common mechanistic link to prosurvival mitochondrial priming. Cells undergoing early adaptive drug escape are in proliferative-metabolic quiescent, with enhanced EMT-ness and stem cell signaling, exhibiting global bioenergetics suppression including reverse Warburg, and are susceptible to glutamine deprivation and TGFβ2 inhibition. Our study further supports a preemptive therapeutic targeting of bioenergetics and mitochondrial priming to impact early drug-escape emergence using EGFR precision inhibitor combined with broad BH3-mimetic to interrupt BCL-2/BCL-xL together, but not BCL-2 alone.

Airway remodeling is strongly correlated with the progression of chronic obstructive pulmonary disease (COPD). In this study, our goal was to characterize progressive structural changes in site-specific airways, along with the temporal and spatial expression of transforming growth factor (TGF)-β in the lungs of male spontaneously hypertensive rats exposed to tobacco smoke (TS). Our studies demonstrated that TS-induced changes of the airways is dependent on airway generation and exposure duration for proximal, midlevel, and distal airways. Stratified squamous epithelial cell metaplasia was evident in the most proximal airways after 4 and 12 weeks but with minimal levels of TGF-β-positive epithelial cells after only 4 weeks of exposure. In contrast, epithelial cells in midlevel and distal airways were strongly TGF-β positive at both 4 and 12 weeks of TS exposure. Airway smooth muscle volume increased significantly at 4 and 12 weeks in midlevel airways. Immunohistochemistry of TGF-β was also found to be significantly increased at 4 and 12 weeks in lymphoid tissues and alveolar macrophages. ELISA of whole-lung homogenate demonstrated that TGF-β2 was increased after 4 and 12 weeks of TS exposure, whereas TGF-β1 was decreased at 12 weeks of TS exposure. Airway levels of messenger RNA for TGF-β2, as well as platelet-derived growth factor-A, granulocyte-macrophage colony-stimulating factor, and vascular endothelial growth factor-α, growth factors regulated by TGF-β, were significantly decreased in animals after 12 weeks of TS exposure. Our data indicate that TS increases TGF-β in epithelial and inflammatory cells in connection with airway remodeling, although the specific role of each TGF-β isoform remains to be defined in TS-induced airway injury and disease.

Background: Lung fibroblasts play a central role in maintaining lung homeostasis and facilitating repair through the synthesis and organization of the extracellular matrix (ECM). This study investigated the cross-talk between interleukin-1 alpha (IL-1α) and transforming growth factor-β (TGF-β) signaling, two key regulators in tissue repair and fibrosis, in the context of lung fibroblast repair in the healthy lung., Results: Stimulation of lung fibroblasts with TGF-β1 and TGF-β2 induced collagen-I and fibronectin protein expression (p < 0.05), a response inhibited with co-treatment with IL-1α (p < 0.05). Additionally, TGF-β1 and TGF-β2 induced myofibroblast differentiation, and collagen-I gel contraction, which were both suppressed by IL-1α (p < 0.05). In contrast, interleukin (IL)-6, IL-8 and thymic stromal lymphopoietin induced by IL-1α, were unaffected by TGF-β1 or TGF-β2. Mechanistically, IL-1α administration led to the suppression of TGF-β1 and TGF-β2 signaling, through downregulation of mRNA and protein for TGF-β receptor II and the downstream adaptor protein TRAF6, but not through miR-146a that is known to be induced by IL-1α., Discussion: IL-1α acts as a master regulator, modulating TGF-β1 and TGF-β2-induced ECM production, remodeling, and myofibroblast differentiation in human lung fibroblasts, playing a vital role in balancing tissue repair versus fibrosis. Further research is required to understand the dysregulated cross-talk between IL-1α and TGF-β signaling in chronic lung diseases and the exploration of therapeutic opportunities., Methods: Primary human lung fibroblasts (PHLF) were treated with media control, or 1 ng/ml IL-1α with or without 50 ng/ml TGF-β1 or TGF-β2 for 1, 6 and 72 h. Cell lysates were assessed for the expression of ECM proteins and signaling molecules by western blot, miRNA by qPCR, mRNA by RNA sequencing and cell supernatants for cytokine production by ELISA. PHLFs were also seeded in non-tethered collagen-I gels to measure contraction, and myofibroblast differentiation using confocal microscopy., Competing Interests: Declaration of competing interest The authors declare no competing financial disclosures., (Copyright © 2024. Published by Elsevier B.V.)

Coronary artery aneurysms (CAA) remain an important complication of Kawasaki disease (KD), the most common form of pediatric acquired heart disease in developed countries. Potentially life-threatening CAA develop in 25% of untreated children and 5% of children treated with high-dose intravenous immunoglobulin during the acute phase of the self-limited vasculitis. Noncoronary artery aneurysms (NCAA) in extraparenchymal, muscular arteries occur in a minority of patients with KD who also have CAA, yet little is understood about their formation and remodeling. We postulated that activation of the transforming growth factor-β (TGF-β) pathway in KD may influence formation and remodeling of aneurysms in iliac, femoral, and axillary arteries, the most common sites for NCAA. We studied a resected axillary artery from one adult and endarterectomy tissue from the femoral artery from a second adult, both with a history of CAA and NCAA following KD in infancy. Histology of the axillary artery aneurysm revealed destruction of the internal elastic lamina and recanalization of organized thrombus, while the endarterectomy specimen showed dense calcification and luminal myofibroblastic proliferation. Immunohistochemistry for molecules in the TGF-β signaling pathway revealed increased expression of TGF-β2, TGF-β receptor 2, and phosphorylated SMAD3. These findings suggest ongoing tissue remodeling of the aneurysms decades after the acute injury and demonstrate the importance of the TGF-β signaling pathway in this process.

BackgroundRELM-β has been implicated in airways inflammation and remodelling in murine models. Its possible functions in human airways are largely unknown. The aim was to address the hypothesis that RELM-β plays a role in extracellular matrix deposition in asthmatic airways.MethodsThe effects of RELM-β gene deficiency were studied in a model of allergen exposure in mice sensitised and challenged with Aspergillus fumigatus (Af). RELM-β expression was investigated in bronchial biopsies from asthmatic patients. Direct regulatory effects of RELM-β on human lung fibroblasts were examined using primary cultures and the MRC5 cell line in vitro.ResultsSensitisation and challenge of wild-type mice with Af-induced release of RELM-β with a time course coincident with that of procollagen in the airways. Af-induced expression of mRNA encoding some, but not all ECM in the lung parenchyma was attenuated in RELM-β-/- mice. RELM-β expression was significantly increased in the bronchial submucosa of human asthmatics compared with controls, and its expression correlated positively with that of fibronectin and α-smooth muscle actin. In addition to epithelial cells, macrophages, fibroblasts and vascular endothelial cells formed the majority of cells expressing RELM-β in the submucosa. Exposure to RELM-β increased TGF-β1, TGF-β2, collagen I, fibronectin, smooth muscle α-actin, laminin α1, and hyaluronan and proteoglycan link protein 1 (Hapl1) production as well as proliferation by human lung fibroblasts in vitro. These changes were associated with activation of ERK1/2 in MRC5 cells.ConclusionThe data are consistent with the hypothesis that elevated RELM-β expression in asthmatic airways contributes to airways remodelling at least partly by increasing fibroblast proliferation and differentiation with resulting deposition of extracellular matrix proteins.

Using a viral model of the demyelinating disease multiple sclerosis (MS), we show that intraspinal transplantation of human embryonic stem cell-derived neural precursor cells (hNPCs) results in sustained clinical recovery, although hNPCs were not detectable beyond day 8 posttransplantation. Improved motor skills were associated with a reduction in neuroinflammation, decreased demyelination, and enhanced remyelination. Evidence indicates that the reduced neuroinflammation is correlated with an increased number of CD4(+)CD25(+)FOXP3(+) regulatory T cells (Tregs) within the spinal cords. Coculture of hNPCs with activated T cells resulted in reduced T cell proliferation and increased Treg numbers. The hNPCs acted, in part, through secretion of TGF-β1 and TGF-β2. These findings indicate that the transient presence of hNPCs transplanted in an animal model of MS has powerful immunomodulatory effects and mediates recovery. Further investigation of the restorative effects of hNPC transplantation may aid in the development of clinically relevant MS treatments.

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The extracellular environment possesses a rich milieu of biophysical and biochemical signaling cues that are simultaneously integrated by cells and influence cellular phenotype. Yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (WWTR1; TAZ), two important signaling molecules of the Hippo pathway, have been recently implicated as nuclear relays of cytoskeletal changes mediated by substratum rigidity and topography. These proteins intersect with other important intracellular signaling pathways (e.g. Wnt and TGFβ). In the cornea, epithelial cells adhere to the stroma through a 3-dimensional topography-rich basement membrane, with features in the nano-submicron size-scale that are capable of profoundly modulating a wide range of fundamental cell behaviors. The influences of substratum-topography, YAP/TAZ knockdown, and HSP90 inhibition on cell morphology, YAP/TAZ localization, and the expression of TGFβ2 and CTGF, were investigated. The results demonstrate (a) that knockdown of TAZ enhances contact guidance in a YAP dependent manner, (b) that CTGF is predominantly regulated by YAP and not TAZ, and (c) that TGFβ2 is regulated by both YAP and TAZ in these cells. Additionally, inhibition of HSP90 resulted in nuclear localization and subsequent transcriptional-activation of YAP, formation of cell-cell junctions and co-localization of E-cadherin and β-catenin at adherens junctions. Results presented in this study reflect the complexities underlying the molecular relationships between the cytoskeleton, growth factors, heat shock proteins, and co-activators of transcription that impact mechanotransduction. The data reveal the importance of YAP/TAZ on the cell behaviors, and gene and protein expression.

BackgroundNeural crest defects lead to congenital heart disease involving outflow tract malformation. Integrin-linked-kinase (ILK) plays important roles in multiple cellular processes and embryogenesis. ILK is expressed in the neural crest, but its role in neural crest and outflow tract morphogenesis remains unknown.ResultsWe ablated ILK specifically in the neural crest using the Wnt1-Cre transgene. ILK ablation resulted in abnormal migration and overpopulation of neural crest cells in the pharyngeal arches and outflow tract and a significant reduction in the expression of neural cell adhesion molecule (NCAM) and extracellular matrix components. ILK mutant embryos exhibited an enlarged common arterial trunk and ventricular septal defect. Reduced smooth muscle differentiation, but increased ossification and neurogenesis/innervation were observed in ILK mutant outflow tract that may partly be due to reduced transforming growth factor β2 (TGFβ2) but increased bone morphogenetic protein (BMP) signaling. Consistent with these observations, microarray analysis of fluorescence-activated cell sorting (FACS)-sorted neural crest cells revealed reduced expression of genes associated with muscle differentiation, but increased expression of genes of neurogenesis and osteogenesis.ConclusionsOur results demonstrate that ILK plays essential roles in neural crest and outflow tract development by mediating complex crosstalk between cell matrix and multiple signaling pathways. Changes in these pathways may collectively result in the unique neural crest and outflow tract phenotypes observed in ILK mutants.

Histone deacetylase 3 (HDAC3) plays a critical role in the maintenance of endothelial integrity and other physiological processes. In this study, we demonstrated that HDAC3 undergoes unconventional splicing during stem cell differentiation. Four different splicing variants have been identified, designated as HD3α, -β, -γ, and -δ, respectively. HD3α was confirmed in stem cell differentiation by specific antibody against the sequences from intron 12. Immunofluorescence staining indicated that the HD3α isoform co-localized with CD31-positive or α-smooth muscle actin-positive cells at different developmental stages of mouse embryos. Overexpression of HD3α reprogrammed human aortic endothelial cells into mesenchymal cells featuring an endothelial-to-mesenchymal transition (EndMT) phenotype. HD3α directly interacts with HDAC3 and Akt1 and selectively activates transforming growth factor β2 (TGFβ2) secretion and cleavage. TGFβ2 functioned as an autocrine and/or paracrine EndMT factor. The HD3α-induced EndMT was both PI3K/Akt- and TGFβ2-dependent. This study provides the first evidence of the role of HDAC3 splicing in the maintenance of endothelial integrity.

Islet transplantation has proven to be a successful strategy to restore normoglycemia in patients with type 1 diabetes (T1D). However, the dearth of cadaveric islets available for transplantation hampers the widespread application of this treatment option. Although human embryonic stem cells and induced pluripotent stem cells are capable of generating insulin-producing cells in vitro when provided with the appropriate inductive cues, the insulin-expressing cells that develop behave more like immature β-cells with minimal sensitivity to glucose stimulation. Here, we identify a set of signaling factors expressed in mouse embryonic mesenchyme during the time when foregut and pancreatic progenitors are specified and test their activities during in vitro differentiation of human embryonic stem cells. Several of the identified factors work in concert to expand the pancreatic progenitor pool. Interestingly, transforming growth factor (TGF)-β ligands, most potent in inducing pancreatic progenitors, display strong inhibitory effects on subsequent endocrine cell differentiation. Treatment with TGF-β ligands, followed by the addition of a TGF-β receptor antagonist, dramatically increased the number of insulin-producing cells in vitro, demonstrating the need for dynamic temporal regulation of TGF-β signaling during in vitro differentiation. These studies illustrate the need to precisely mimic the in vivo conditions to fully recapitulate pancreatic lineage specification in vitro.

Vertebrates have achieved great evolutionary success due in large part to the anatomical diversification of their jaw complex, which allows them to inhabit almost every ecological niche. While many studies have focused on mechanisms that pattern the jaw skeleton, much remains to be understood about the origins of novelty and diversity in the closely associated musculature. To address this issue, we focused on parrots, which have acquired two anatomically unique jaw muscles: the ethmomandibular and the pseudomasseter. In parrot embryos, we observe distinct and highly derived expression patterns for Scx, Bmp4, Tgfβ2 and Six2 in neural crest-derived mesenchyme destined to form jaw muscle connective tissues. Furthermore, immunohistochemical analysis reveals that cell proliferation is more active in the cells within the jaw muscle than in surrounding connective tissue cells. This biased and differentially regulated mode of cell proliferation in cranial musculoskeletal tissues may allow these unusual jaw muscles to extend towards their new attachment sites. We conclude that the alteration of neural crest-derived connective tissue distribution during development may underlie the spatial changes in jaw musculoskeletal architecture found only in parrots. Thus, parrots provide valuable insights into molecular and cellular mechanisms that may generate evolutionary novelties with functionally adaptive significance.

Purpose: To investigate the effects of anterior chamber pigment dispersion on ocular immune privilege and the possible mechanisms involved in a DBA/2J mouse model of pigmentary glaucoma., Methods: DBA/2J mice were utilized as a pigment dispersion model, and age-matched C57BL/6J mice were used as the control group in this study. Proteins in the aqueous humor (AH) and serum were quantified using the bicinchoninic acid assay. Immune cells in the AH were detected using hematoxylin and eosin staining and immunocytochemistry. The expression of TGF-β2 in the AH and cytokine levels (IL-10, IFN-γ) in serum were measured using ELISA. Anterior chamber-associated immune deviation (ACAID) was induced in DBA/2J mice by injecting antigens into the anterior chamber. Delayed-type hypersensitivity (DTH) assays were used to assess the induction of ACAID. In DBA/2J mice, before and after pigment dispersion, following anterior chamber injection of pigment particles, and after ACAID modeling, the expression of regulatory T cells (Tregs) was detected using flow cytometry., Results: Compared to C57BL/6J mice, the protein concentration, immune cell count, and TGF-β2 levels in the AH were elevated in DBA/2J mice. Protein concentration and IL-10 levels in serum were increased, while IFN-γ levels were decreased in DBA/2J. Additionally, the expression of Treg cells in the spleen of DBA/2J mice was significantly increased after pigment dispersion and anterior chamber injection of pigment particles. At 3 and 6 months, DTH responses in DBA/2J mice were not inhibited, thus preventing ACAID induction. However, the opposite was observed at 9 months in DBA/2J mice. Furthermore, the ACAID group exhibited an augmented expression of Treg cells., Conclusions: Dispersion of pigment particles in the anterior chamber of the eye enhances the state of ocular immune privilege by influencing the immunosuppressive microenvironment and inducing more Treg cells to reestablish ACAID.

Two new studies show that haploinsufficiency for TGFB2 causes a familial syndrome of thoracic aortic aneurysms and dissections with other clinical features that overlap the Marfan, Loeys-Dietz spectrum of syndromes. Their finding of loss-of-function mutations in yet another transforming growth factor (TGF)-β pathway gene reinforces the seeming paradox of observed increases in the downstream TGF-β signaling pathway.

Background: To date, the difference in microRNA expression profiles in tears of dry eye patients and healthy people has not been reported. In current study, we evaluated the significance of microRNAs and transforming growth factor beta2 (TGF-β2) in distinguishing dry eye. Methods: A total of 138 patients with dry eye from October 2017 to October 2018 were selected. During the same period, 138 healthy persons were collected. All patients were followed up for 12 months through outpatient, telephone or medical records and the time of corneal injury was recorded. Results: Compared with healthy people, TGF-β2 concentrations in dry eye patients were significantly decreased (P < 0.05). Array analysis, predictive software and dual-luciferase reporter assays showed that miR-450b-5p, miR-1283 and miR-3671 can target TGF-β2 expression. Tear miR-450b-5p, miR-1283 and miR-3671 concentrations were significantly higher in dry eye patients than healthy people. A logistic regression model combining miR-450b-5p, miR-1283, miR-3671 and TGF-β2 was performed. This model presented a high discriminating value (AUC: 0.907, 0.876–0.939, P < 0.001) than any single indicator, and the sensitivity and specificity were 77.7% and 92.7%, respectively. Compared with the low miR-450b-5p, low miR-1283, low miR-3671 and high TGF-β2 groups, the high miR-450b-5p, high miR-1283, high miR-3671 and low TGF-β2 groups had a significantly higher probability of corneal injury (TGF-β2: χ2 = 5.762, P = 0.016; miR-450b-5p: χ2 = 13.267, P < 0.001; miR-1283: χ2 = 19.431, P < 0.001; miR-3671: χ2 = 8.131, P = 0.004). Conclusion: Current model combining tear miR-450b-5p, miR-1283, miR-3671 and TGF-β2 had important values in the identification of dry eye and was of great value in evaluating the risk of corneal injury. [ABSTRACT FROM AUTHOR]

BackgroundAirway narrowing associated with chronic asthma results in the transmission of injurious compressive forces to the bronchial epithelium and promotes the release of pro-inflammatory mediators and the denudation of the bronchial epithelium. While the individual effects of compression or denudation are well characterized, there is no data to elucidate how these cells respond to the application of mechanical compression in the presence of a compromised epithelial layer.MethodsAccordingly, differentiated normal human bronchial epithelial cells were exposed to one of four conditions: 1) unperturbed control cells, 2) single scrape wound only, 3) static compression (6 hours of 30 cmH2O), and 4) 6 hours of static compression after a scrape wound. Following treatment, wound closure rate was recorded, media was assayed for mediator content and the cytoskeletal network was fluorescently labeled.ResultsWe found that mechanical compression and scrape injury increase TGF-beta2 and endothelin-1 secretion, while EGF content in the media is attenuated with both injury modes. The application of compression after a pre-existing scrape wound augmented these observations, and also decreased PGE2 media content. Compression stimulated depolymerization of the actin cytoskeleton and significantly attenuated wound healing. Closure rate was partially restored with the addition of exogenous PGE2, but not EGF.ConclusionOur results suggest that mechanical compression reduces the capacity of the bronchial epithelium to close wounds, and is, in part, mediated by PGE2 and a compromised cytoskeleton.

The epithelium influences the mesenchyme during dynamic processes such as embryogenesis, wound healing, fibrosis, and carcinogenesis. Since transforming growth factor-beta (TGF-beta) modulates these processes, we hypothesized that epithelial-derived TGF-beta also plays a critical role in maintaining the extracellular matrix at basal conditions. We utilized an in vitro model of the epithelial-mesenchymal trophic unit in the human airways to determine the role of epithelial-derived TGF-beta in modulating the extracellular matrix under basal and wound-healing conditions. When differentiated at an air-liquid interface, the human bronchial epithelium produces active TGF-beta2 at a concentration of 50-70 pg/ml, whereas TGF-beta1 is undetectable. TGF-beta2 increases two- to threefold following scrape injury in a dose-dependent fashion and significantly enhances both alpha-smooth muscle actin expression in the underlying collagen-embedded fibroblasts and secretion of tenascin-C into the matrix. Multiphoton microscopy demonstrates substantially enhanced second harmonic generation from fibrillar collagen in the matrix. Pretreatment of the matrix with either sirolimus (2.5 nM) or paclitaxel (10 nM) abolishes the increases in both TGF-beta2 and second harmonic generation in response to epithelial injury. In the absence of the epithelium, exogenous active TGF-beta2 (0-400 pg/ml) produces a biphasic response in the second harmonic signal with a minimum occurring at the epithelial-derived basal level. We conclude that epithelial-derived TGF-beta2 is secreted in response to injury, significantly alters the bulk optical properties of the extracellular matrix, and its tight regulation may be required for normal collagen homeostasis.

The epithelium influences the mesenchyme during dynamic processes such as embryogenesis, wound healing, fibrosis, and carcinogenesis. Since transforming growth factor-beta (TGF-beta) modulates these processes, we hypothesized that epithelial-derived TGF-beta also plays a critical role in maintaining the extracellular matrix at basal conditions. We utilized an in vitro model of the epithelial-mesenchymal trophic unit in the human airways to determine the role of epithelial-derived TGF-beta in modulating the extracellular matrix under basal and wound-healing conditions. When differentiated at an air-liquid interface, the human bronchial epithelium produces active TGF-beta2 at a concentration of 50-70 pg/ml, whereas TGF-beta1 is undetectable. TGF-beta2 increases two- to threefold following scrape injury in a dose-dependent fashion and significantly enhances both alpha-smooth muscle actin expression in the underlying collagen-embedded fibroblasts and secretion of tenascin-C into the matrix. Multiphoton microscopy demonstrates substantially enhanced second harmonic generation from fibrillar collagen in the matrix. Pretreatment of the matrix with either sirolimus (2.5 nM) or paclitaxel (10 nM) abolishes the increases in both TGF-beta2 and second harmonic generation in response to epithelial injury. In the absence of the epithelium, exogenous active TGF-beta2 (0-400 pg/ml) produces a biphasic response in the second harmonic signal with a minimum occurring at the epithelial-derived basal level. We conclude that epithelial-derived TGF-beta2 is secreted in response to injury, significantly alters the bulk optical properties of the extracellular matrix, and its tight regulation may be required for normal collagen homeostasis.

Purpose: The fifth most common cancer is allocated to bladder cancer (BC) worldwide. Understanding the molecular mechanisms of BC invasion and metastasis to identify target therapeutic strategies will improve disease survival. So the aim of this study was to measure expression rate of zinc finger E-box binding homeobox 1 (ZEB1) and transforming growth factor-beta2 (TGF-β2) mRNA in tissue samples of patients with BC and its healthy adjacent tissue samples and their association with muscle invasion, size and grade of the tumor. Materials and Methods: Tissue samples were collected from 35 newly diagnosed untreated patients with BC from 2013 to 2014. Total RNA was extracted from about 50-mg tissue samples using TRIzol reagent. TAKARA SYBR Premix EX Tag II was applied to determine the rate of mRNA expression by real-time polymerase chain reaction (PCR). To obtain final validation, PCR product of ZEB1 and TGF-β2 were sequenced. STATA 11 software was used to analyze the data. Results: The expression level of ZEB1 in tumor samples was significantly more than of in healthy adjacent tissue samples. Up-regulation of TGF-β2 showed a strong association with muscle invasion (p=0.017). There was also demonstrated a relationship between over expression of ZEB1 with the tumor size (p=0.050). Conclusions: It looks ZEB1 and TGF-β2 had a role in BC patients. In this study ZEB1 expression was higher in BC tissues than that of in healthy control tissues. There was demonstrated a markedly association between overexpression of TGF-β2 and muscle invasion. Therefore, they are supposed to be candidate as potential biomarkers for early detection and progression of BC.

Calcitriol levels increase during pregnancy, contributing to the hormonal and immunological balance, but its deficiency has been associated with problems during this period. Meanwhile, transforming growth factors-β (TGF-βs) play an important role in the maintenance of fetal-maternal immune tolerance; however, exacerbated concentrations of this growth factor are associated with complicated pregnancies. Therefore, we studied the effects of calcitriol on TGF-βs and their receptors in trophoblast cells. Term placentas from uncomplicated pregnancies after cesarean sections were used for cell cultures. Basal gene expression and the effect of calcitriol upon TGF-β1, TGF-β2, TGF-β3, and their receptors TGF-βR1 and TGF-βR2 were assessed using real-time PCR from trophoblast cells. The presence of TGF-β1, 2, 3, and TGF-βR1 were evaluated by immunofluorescence, and the protein abundance and secretion of TGF-β1 were assessed by Western blot and ELISA, respectively. Basal gene expression of TGF-β1 in trophoblast from term placentas was higher than TGF-β2 and TGF-β3, while TGF-βR2 was higher than TGF-βR1. The presence and cellular localization of TGF-β1, 2, 3, and TGF-βR1 were detected in the cytoplasm of syncytiotrophoblast, with TGF-β1 showing the highest intensity. Calcitriol significantly inhibited gene expression of TGF-β1, TGF-β2, and TGF-βR1. Likewise, calcitriol decreased the secretion and abundance of TGF-β1. In conclusion, results indicate that calcitriol is a regulator of TGF-βs in cultured trophoblast cells from term placentas and therefore may be an important player in the development of healthy pregnancies., Competing Interests: Declaration of Competing Interest none., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Human cartilage tissue remains a challenge for the development of therapeutic options due to its poor vascularization and reduced regenerative capacities. There are a variety of research approaches dealing with cartilage tissue engineering. In addition to different biomaterials, numerous cell populations have been investigated in bioreactor-supported experimental setups to improve cartilage tissue engineering. The concept of the present study was to investigate spider silk cocoons as scaffold seeded with adipose-derived stromal cells (ASC) in a custom-made bioreactor model using cyclic axial compression to engineer cartilage-like tissue. For chemical induction of differentiation, BMP-7 and TGF-β2 were added and changes in cell morphology and de-novo tissue formation were investigated using histological staining to verify chondrogenic differentiation. By seeding spider silk cocoons with ASC, a high colonization density and cell proliferation could be achieved. Mechanical induction of differentiation using a newly established bioreactor model led to a more roundish cell phenotype and new extracellular matrix formation, indicating a chondrogenic differentiation. The addition of BMP-7 and TGF-β2 enhanced the expression of cartilage specific markers in immunohistochemical staining. Overall, the present study can be seen as pilot study and valuable complementation to the published literature., Competing Interests: Declaration of conflicting interestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

MicroRNAs are involved in the immune systems of host animals and play essential roles in several immune-related pathways. In the current study, we investigated the systemic biological function of the chicken miRNA gga-miR-148a-3p on immune responses in chicken lines resistant and susceptible to HPAIV-H5N1. We found that gga-miR-148a expression in the lung tissue of H5N1-resistant chickens was significantly downregulated during HPAIV-H5N1 infection. Overexpression of gga-miR-148a and a reporter construct with wild type or mutant IFN-γ, MAPK11, and TGF-β2 3' untranslated region (3' UTR)-luciferase in chicken fibroblasts showed that gga-miR-148a acted as a direct translational repressor of IFN-γ, MAPK11, and TGF-β2 by targeting their 3' UTRs. Furthermore, miR-148a directly and negatively influenced the expression of signalling molecules related to the MAPK signalling pathway, including MAPK11, TGF-β2, and Jun, and regulated antiviral responses through interferon-stimulated genes and MHC class I and class II genes by targeting IFN-γ. Downstream of the MAPK signalling pathway, several proinflammatory cytokines such as IL-1β, IFN-γ, IL-6, TNF-α, IFN-β, and interferon-stimulated genes were downregulated by the overexpression of gga-miR-148a. Our data suggest that gga-miR-148a-3p is an important regulator of the MAPK signalling pathway and antiviral response. These findings improve our understanding of the biological functions of gga-miR-148a-3p, the mechanisms underlying the MAPK signalling pathway, and the antiviral response to HPAIV-H5N1 infection in chickens as well as the role of gga-miR-148a-3p in improving the overall performance of chicken immune responses for breeding disease-resistant chickens., (© 2023. The Author(s).)

BACKGROUND: It is revealed from the cell and gene level that bone morphogenetic protein 2 (BMP2) and transforming growth factor β2 (TGFβ2) synergistically promote the osteogenic differentiation of bone marrow mesenchymal stem cells, providing an experimental and theoretical basis for the bone regeneration. OBJECTIVE: To investigate the overexpression of BMP2 and TGFβ2 in vitro to synergistically promote the osteogenic differentiation of bone marrow mesenchymal stem cells. METHODS: Primary rat bone marrow mesenchymal stem cells were obtained by enzymatic digestion. Recombinant plasmids of BMP2 and TGFβ2 were packaged by lentivirus. The lentivirus carrying the recombinant plasmids of the target genes BMP2 and TGFβ2 was transfected into the third generation of bone marrow mesenchymal stem cells. MTS (cell proliferation assay) was used to detect the proliferation of bone marrow mesenchymal stem cells in different treatment groups at different time points. Quantitative PCR was used to detect the overexpression of BMP2 and TGFβ2 at mRNA levels. Immunohistochemistry of cell slides was performed to detect the expression level of osteogenic differentiation marker RUNX2 at the protein level under two-dimensional mixed culture conditions. The cell pellet obtained by three-dimensional culture in vitro was subjected to paraffin sectioning for Vonkossa staining (silver staining) to determine the positive region and expression intensity of osteogenic differentiation, and quantitative analysis was performed. RESULTS AND CONCLUSION: (1) The proliferation of bone marrow mesenchymal stem cells transfected with BMP2 and TGFβ2 was not statistically different from that of untransfected cells (P > 0.05). (2) The BMP2 and TGFβ2 mRNA levels were significantly higher in the transfected cells than untransfected ones (P < 0.05). (3) RUNX2 immunohistochemistry under the in vitro two-dimensional culture conditions and Vonkossa staining under the in vitro three-dimensional culture showed that the BMP2 and TGFβ2 co-transfected bone marrow mesenchymal stem cells had stronger osteogenic ability than BMP2 or TGFβ2 alone transfected cells (P < 0.05). In conclusion, the overexpression of recombinant plasmids of BMP2 and TGFβ2 carried by lentivirus synergistically promotes the osteogenic differentiation of rat bone marrow mesenchymal stem cells. [ABSTRACT FROM AUTHOR]