TGF-β1 Exposure Research Articles

Survival of periodontal ligament myofibroblasts after short-term mechanical strain in rats and in vitro: Could myofibroblasts contribute to orthodontic relapse?

To investigate in vivo whether myofibroblasts formed in the PDL after exposure to short-term high experimental orthodontic forces in rats survive. To study in vitro whether human PDL fibroblasts can differentiate into myofibroblasts and survive when chemical or mechanical stimuli are removed. Nine 6-week-old male Wistar rats were used in this experiment. Rat molars were exposed to high but rapidly decreasing experimental orthodontic forces by applying a rubber band and analyzed for the presence of myofibroblasts using ASMA staining. In vitro, human periodontal ligament (PDL) fibroblasts were exposed to transforming growth factor β1 (TGFβ1) and/or mechanical stress and monitored for myofibroblast formation and survival after these stimuli were abrogated. In vivo exposure to orthodontic forces strongly induced myofibroblast formation in the stretched regions of the PDL. Furthermore, many PDL myofibroblasts remained present 6 days after exposure to these short-term high orthodontic forces. Human PDL fibroblasts were shown to differentiate into myofibroblasts after 2 days of TGFβ1 exposure and survive for at least 2 more days after removing chemical stimuli (TGFβ1) or mechanical strain. Under in vitro conditions, both TGFβ1 and mechanical strain for 3 days promoted (myo)fibroblast formation, and these cells persisted for 3 more days after the removal of both stimuli. PDL myofibroblasts survive after the removal of mechanical strain in vivo and in vitro. This supports the hypothesis that myofibroblasts, which form in response to mechanical strain and chemical cues in the periodontal ligament (PDL), play a role in relapse following orthodontic tooth movement.

Unraveling the Exosome-miR-133a Axis: Targeting TGF-β Signaling via WJ-MSC-Derived Exosomes for Anti-Fibrotic Therapy in Liver Fibrosis.

One of the primary drivers of liver fibrosis is the excessive accumulation of ECM, primarily caused by the over-proliferation of HSCs. The activation of HSCs by TGF-β has a critical role in initiating fibrosis. Recent studies have suggested that miRNA-133a significantly regulates the fibrogenesis process, which its downregulation is associated with the fibrosis progression. Understanding the role of miRNA-133a provides potential therapeutic insights for targeting TGF-β signaling and mitigating liver fibrosis. We investigated whether exosomes could attenuate liver fibrosis by enhancing the antifibrotic effects of miR-133a. The LX-2 cell line was treated with TGF-β for 24 hours, followed by an additional 24 hours of treatment with exosomes. After this treatment period, we assessed the mRNA expression levels of α-SMA, collagen 1, and miR-133a, as well as the protein levels of p-Smad3. TGF-β exposure significantly increased the expression level of α-SMA and collagen 1 genes and elevated the levels of p-Smad3 protein. Additionally, it resulted in a significant downregulation of miR-133a compared to the control group. Exosome administration effectively reduced the TGF-β-induced upregulation of p-Smad3, α-SMA, and collagen 1 genes, but increased miR-133a expression levels. Our findings indicate that by partially mitigating the downregulation of miR-133a, exosomes can effectively inhibit the persistent activation of HSCs. Furthermore, in the context of in vitro liver fibrosis, exosomes can suppress the TGF-β/Smad3 pathway, reducing the accumulation of ECM.

Cell mediated reactions create TGF-β delivery limitations in engineered cartilage

During native cartilage development, endogenous TGF-β activity is tightly regulated by cell-mediated chemical reactions in the extracellular milieu (e.g., matrix and receptor binding), providing spatiotemporal control in a manner that is localized and short acting. These regulatory paradigms appear to be at odds with TGF-β delivery needs in tissue engineering (TE) where administered TGF-β is required to transport long distances or reside in tissues for extended durations. In this study, we perform a novel examination of the influence of cell-mediated reactions on the spatiotemporal distribution of administered TGF-β in cartilage TE applications. Reaction rates of TGF-β binding to cell-deposited ECM and TGF-β internalization by cell receptors are experimentally characterized in bovine chondrocyte-seeded tissue constructs. TGF-β binding to the construct ECM exhibits non-linear Brunauer–Emmett–Teller (BET) adsorption behavior, indicating that as many as seven TGF-β molecules can aggregate at a binding site. Cell-mediated TGF-β internalization rates exhibit a biphasic trend, following a Michaelis–Menten relation (Vmax = 2.4 molecules cell-1 s-1, Km = 1.7 ng mL-1) at low ligand doses (≤130 ng/mL), but exhibit an unanticipated non-saturating power trend at higher doses (≥130 ng/mL). Computational models are developed to illustrate the influence of these reactions on TGF-β spatiotemporal delivery profiles for conventional TGF-β administration platforms. For TGF-β delivery via supplementation in culture medium, these reactions give rise to pronounced steady state TGF-β spatial gradients; TGF-β concentration decays by ∼90 % at a depth of only 500 μm from the media-exposed surface. For TGF-β delivery via heparin-conjugated affinity scaffolds, cell mediated internalization reactions significantly reduce the TGF-β scaffold retention time (160–360-fold reduction) relative to acellular heparin scaffolds. This work establishes the significant limitations that cell-mediated chemical reactions engender for TGF-β delivery and highlights the need for novel delivery platforms that account for these reactions to achieve optimal TGF-β exposure profiles. Statement of significanceDuring native cartilage development, endogenous TGF-β activity is tightly regulated by cell-mediated chemical reactions in the extracellular milieu (e.g., matrix and receptor binding), providing spatiotemporal control in a manner that is localized and short acting. However, the effect of these reactions on the delivery of exogenous TGF-β to engineered cartilage tissues remains not well understood. In this study, we demonstrate that cell-mediated reactions significantly restrict the delivery of TGF-β to cells in engineered cartilage tissue constructs. For delivery via media supplementation, reactions significantly limit TGF-β penetration into constructs. For delivery via scaffold loading, reactions significantly limit TGF-β residence time in constructs. Overall, these results illustrate the impact of cell-mediated chemical reactions on TGF-β delivery profiles and support the importance of accounting for these reactions when designing TGF-β delivery platforms for promoting cartilage regeneration.

MiR-335-3p attenuates transforming growth factor beta 1-induced fibrosis by suppressing Thrombospondin 1.

Pulmonary fibrosis is characterized by excessive extracellular matrix (ECM) accumulation caused by detrimental stimuli. The progressive impairment in lung functions is chronic and highly fatal, presenting itself as a global health challenge. Because of the lack of efficacious treatments, the underlying mechanism should be investigated. The progression of fibrosis involves transforming growth factor-beta 1 (TGF-β1), which accelerates ECM production via epithelial-mesenchymal transition and cell invasion. As microRNAs (miRNAs) serve as regulators of disease development and progression, this study aimed to investigate the interaction of miRNAs and target genes that could contribute to pulmonary fibrosis when exposed to TGF-β1. Differentially expressed mRNA and miRNA were identified in respiratory epithelial cells via transcriptome analysis by using the constructed TGF-β1-induced fibrosis model. Our results revealed a significant increase in the expression of thrombospondin 1 (THBS1), which participates in TGF-β1 activation, where THBS1 was identified as a core gene in protein interactions analyzed through bioinformatics. The expression of miR-335-3p, which targets 3'-UTR of THBS1, substantially decreased upon TGF-β1 treatment. The TGF-β1 downstream signal was suppressed by inhibiting the interaction between TGF-β1 and THBS1, consequently alleviating fibrosis. When the miR-335-3p mimic was transfected in TGF-β1-treated respiratory epithelial cells, THBS1 and fibrosis markers were downregulated, while the introduction of miR-335-3p inhibitor exhibited a reverse phenomenon. Our findings demonstrated that TGF-β1 exposure to respiratory epithelial cells led to a decrease in miR-335-3p expression, resulting in the upregulation of THBS1 and ultimately exacerbating fibrosis. This study provides insights into TGF-β1-induced pulmonary fibrosis, suggesting new therapeutic targets and mechanisms.

Increased peritoneal TGF-β1 is associated with ascites-induced NK-cell dysfunction and reduced survival in high-grade epithelial ovarian cancer.

Natural killer (NK) cell therapy represents an attractive immunotherapy approach against recurrent epithelial ovarian cancer (EOC), as EOC is sensitive to NK cell-mediated cytotoxicity. However, NK cell antitumor activity is dampened by suppressive factors in EOC patient ascites. Here, we integrated functional assays, soluble factor analysis, high-dimensional flow cytometry cellular component data and clinical parameters of advanced EOC patients to study the mechanisms of ascites-induced inhibition of NK cells. Using a suppression assay, we found that ascites from EOC patients strongly inhibits peripheral blood-derived NK cells and CD34+ progenitor-derived NK cells, albeit the latter were more resistant. Interestingly, we found that higher ascites-induced NK cell inhibition correlated with reduced progression-free and overall survival in EOC patients. Furthermore, we identified transforming growth factor (TGF)-β1 to correlate with ascites-induced NK cell dysfunction and reduced patient survival. In functional assays, we showed that proliferation and anti-tumor reactivity of CD34+ progenitor-derived NK cells are significantly affected by TGF-β1 exposure. Moreover, inhibition of TGF-β1 signaling with galunisertib partly restored NK cell functionality in some donors. For the cellular components, we showed that the secretome is associated with a different composition of CD45+ cells between ascites of EOC and benign reference samples with higher proportions of macrophages in the EOC patient samples. Furthermore, we revealed that higher TGF-β1 levels are associated with the presence of M2-like macrophages, B cell populations and T-regulatory cells in EOC patient ascites. These findings reveal that targeting TGF-β1 signaling could increase NK cell immune responses in high-grade EOC patients.

TGF-β1 Induces Endoplasmic Reticulum Stress-Dependent Apoptosis in Human Placental Mesenchymal Stem Cells of Fetal Origin through PERK Signaling Pathway

Background: Mesenchymal Stem Cells (MSCs) are pivotal in immunomodulation, hematopoiesis, and tissue repair. The interplay between MSCs and the pathological microenvironment influences their proliferation and differentiation. Transforming Growth Factor-Beta 1 (TGF- β1) serves as a key cytokine in the MSC microenvironment. This study aimed to scrutinize the impact of TGF-β1 on human placenta-derived MSCs of fetal origin (fPMSCs) and elucidate its underlying mechanism. Methods: fPMSCs were isolated, and surface markers were identified by flow cytometry. Cell proliferation in fPMSCs was assessed using Cell Counting Kit-8 (CCK-8) and 5-Ethynyl-2’-Deoxy Uridine (EdU). Apoptosis was detected via Annexin V/PI staining, and apoptosis-related proteins were detected by western blot. Endoplasmic reticulum (ER) stress-related proteins were detected by western blot, and Flou-4 AM staining was utilized to assess intracellular Ca2+ levels under TGF-β1 exposure. The impact of 4-PBA treatment on ER stress and apoptosis was assessed by western blot and Annexin V/PI staining. Additionally, the PERK and p-PERK expressions were evaluated via Western blot. Results: CCK-8 and EdU assays revealed inhibited proliferation of fPMSCs under TGF-β1 exposure. Annexin V/PI staining demonstrated a significant induction of apoptosis in fPMSCs following TGF-β1 treatment. Furthermore, TGF-β1 treatment significantly elevated intracellular Ca2+ levels and the expressions of GRP78, p-eIF2α, and CHOP. Interruption of ER stress with 4-PBA mitigated TGF-β1-induced apoptosis in fPMSCs. Moreover, TGF-β1 increased p-PERK expression. Inhibition of PERK autophosphorylation with GSK2606414 suppressed TGF-β1-induced apoptosis and ER stress in fPMSCs. Conclusion: Our findings indicated that TGF-β1 induced ER stress-dependent apoptosis in fPMSCs through the PERK signaling pathway. These results offer insights into enhancing the therapeutic efficacy of fPMSCs by modulating TGF-β1-induced apoptosis.

TGFβ signaling pathway is altered by HLA-B27 expression, resulting in pathogenic consequences relevant for spondyloarthritis

BackgroundAssociation of HLA-B27 with spondyloarthritis (SpA) has been known for 50 years, but still remains unexplained. We recently showed that HLA-B27 expressed in wing imaginal disc from HLA-B27/human-β2 microglobulin (hβ2m) transgenic Drosophila deregulated bone morphogenetic protein (BMP) pathway by interacting physically with type I BMP receptor (BMPR1) Saxophone (Sax), leading to crossveinless phenotype.MethodsGenetic interaction was studied between activin/transforming growth factor β (TGFβ) pathway and HLA-B27/hβ2m in transgenic Drosophila wings. The HLA-B27-bound peptidome was characterized in wing imaginal discs. In mesenteric lymph node (mLN) T cells from HLA-B27/hβ2m rat (B27 rat), physical interaction between HLA-B27 and activin receptor-like kinase-2 (ALK2), ALK3 and ALK5 BMPR1s, phosphorylation of small mothers against decapentaplegic (SMADs) and proteins of the non-canonical BMP/TGFβ pathways induced by its ligands, and the transcript level of target genes of the TGFβ pathway, were evaluated.ResultsIn HLA-B27/hβ2m transgenic Drosophila, inappropriate signalling through the activin/TGFβ pathway, involving Baboon (Babo), the type I activin/TGFβ receptor, contributed to the crossveinless phenotype, in addition to deregulated BMP pathway. We identified peptides bound to HLA-B27 with the canonical binding motif in HLA-B27/hβ2m transgenic Drosophila wing imaginal disc. We demonstrated specific physical interaction, between HLA-B27/hβ2m and mammalian orthologs of Sax and Babo, i.e. ALK2 and ALK5 (i.e. TGFβ receptor I), in the mLN cells from B27 rat. The magnitude of phosphorylation of SMAD2/3 in response to TGFβ1 was increased in T cells from B27 rats, showing evidence for deregulated TGFβ pathway. Accordingly, expression of several target genes of the pathway was increased in T cells from B27 rats, in basal conditions and/or after TGFβ exposure, including Foxp3, Rorc, Runx1 and Maf. Interestingly, Tgfb1 expression was reduced in naive T cells from B27 rats, even premorbid, an observation consistent with a pro-inflammatory pattern.ConclusionsThis study shows that HLA-B27 alters the TGFβ pathways in Drosophila and B27 rat. Given the importance of this pathway in CD4 + T cells differentiation and regulation, its disturbance could contribute to the abnormal expansion of pro-inflammatory T helper 17 cells and altered regulatory T cell phenotype observed in B27 rats.

Inhibition of the rapamycin-insensitive mTORC1 /4E-BP1 axis attenuates TGF-β1-induced fibrotic response in human Tenon's fibroblasts

Subconjunctival fibrosis is the major cause of failure in both conventional and modern minimally invasive glaucoma surgeries (MIGSs) with subconjunctival filtration. The search for safe and effective anti-fibrotic agents is critical for improving long-term surgical outcomes. In this study, we investigated the effect of inhibiting the rapamycin-insensitive mTORC1/4E-BP1 axis on the transforming growth factor-beta 1(TGF-β1)-induced fibrotic responses in human Tenon's fibroblasts (HTFs), as well as in a rat model of glaucoma filtration surgery (GFS). Primary cultured HTFs were treated with 3 ng/mL TGF-β1 for 24 h, followed by treatment with 10 μM CZ415 for additional 24 h. Rapamycin (10 μM) was utilized as a control for mTORC1/4E-BP1 signaling insensitivity. The expression levels of fibrosis-associated molecules were measured using quantitative real-time PCR, Western blotting, and immunofluorescence analysis. Cell migration was assessed through the scratch wound assay. Additionally, a rat model of GFS was employed to evaluate the anti-fibrotic effect of CZ415 in vivo. Our findings indicated that both rapamycin and CZ415 treatment significantly reduced the TGF-β1-induced cell proliferation, migration, and the expression of pro-fibrotic factors in HTFs. CZ415 also more effectively inhibited TGF-β1-mediated collagen synthesis in HTFs compared to rapamycin. Activation of mTORC1/4E-BP signaling following TGF-β1 exposure was highly suppressed by CZ415 but was only modestly inhibited by rapamycin. Furthermore, CZ415 was found to decrease subconjunctival collagen deposition in rats post GFS. Our results suggest that rapamycin-insensitive mTORC1/4E-BP1 signaling plays a critical role in TGF-β1-driven collagen synthesis in HTFs. This study demonstrated that inhibition of the mTORC1/4E-BP1 axis offers superior anti-fibrotic efficacy compared to rapamycin and represents a promising target for improving the success rate of both traditional and modern GFSs.

Activin A Contribution to Diabetic Kidney Disease Injury and Effects of Activin A Antagonism In-Vitro

Diabetic kidney disease (DKD) is the worldwide leader in end-stage kidney failure and has limited treatment options. Cellular senescence abundance and inflammation contribute to DKD pathogenesis. We previously demonstrated that activin A, an inflammatory mediator of pro-fibrotic kidney injury and cellular senescence, is increased in DKD and correlates with DKD injury and kidney function. We hypothesized that activin A contributes to DKD injury causing pro-fibrotic, pro-inflammatory, and cellular senescence effects in vitro, and that antagonism of activin A is achieved with follistatin. An in vitro model of DKD injury was applied with high-glucose (HG) and transforming growth factor-β (TGFβ) exposure for 24 hours in U-937-derived macrophages and renal epithelial (HK2) cells. Prior to exposure, U-937 cells were converted to M0 macrophages with phorbol myristate acetate (PMA). To examine the potential of reducing activin A release by DKD injured cells, follistatin, an activin A antagonist, was added to the media of cells at the time of injury for 24 hours. Additionally, an activin A neutralizing antibody was added to the collected supernatant of DKD injured cells. Activin A, senescence (p16), pro-fibrotic (collagen I), and pro-inflammatory (monocyte chemoattractant protein [MCP]-1, tumor necrosis factor [TNF]-alpha, interleukin [IL]-6) markers were measured through qPCR, ELISA (activin A), and immunofluorescence staining (activin A). DKD injury in renal epithelial cells increased protein expression of activin A, increased gene expression of activin A, p16, and collagen I, but did not significantly increase MCP-1 expression at 24 hour exposure (Figure 1). Furthermore, DKD injury led to increased activin A release in conditioned medium vs. control renal epithelial cells. Administration of follistatin reduced activin A release by renal epithelial cells. Application of neutralizing antibody reduced activin A levels in collected medium after cell injury. After DKD injury in U-937 derived macrophages, activin A gene expression was increased. Injured macrophages had increased gene expression of TNF-alpha and IL-6, demonstrating a pro-inflammatory phenotype. In conclusion, diabetic kidney injury increases activin A release, inflammation markers, and senescence markers in kidney cells and macrophages, therein contributing to downstream effects that further propagate disease. As shown, follistatin antagonism reduces activin A release by injured cells. Additional studies are needed to explore activin A as a promising therapeutic target and follistatin as a possible treatment in DKD. NIH 5TL1TR002380-07, AG076537, DK123492, DK109134. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Effect of Benidipine Alone and in Combination With Bosentan and Sildenafil in Amelioration of Pulmonary Arterial Hypertension in Experimental Model in Rats.

Pulmonary arterial hypertension (PAH) is a persistent condition affecting the pulmonary arteries' endothelium. Benidipine, a calcium channel blocker, possesses vasodilatory, anti-inflammatory activity, reduces oxidative stress, and inhibits the activity of Transforming growth factor-β (TGF-β) and α-smooth muscle actin (α-SMA). The present study was designed to investigate the effect of benidipine alone and in combination with bosentan and sildenafil on monocrotaline (MCT)-induced pulmonary hypertension in a rat model. PAH was induced by a single-dose administration of MCT in rats. Animals were randomized into different groups and treated with benidipine alone and in combination with bosentan or sildenafil. Various parameters such as hemodynamic parameters, Fulton's index and oxidative stress parameters were performed. Additionally, histopathology of lung and right ventricular of heart tissue, immunohistochemistry, expression of α-SMA, endothelial nitric oxide synthase (eNOS), TGF-β, and RT-PCR, and an in vitro study using human umbilical vein endothelial cells (HUVECs) was also carried out. Treatment of benidipine and its combination exhibited better prevention in the elevated right ventricular systolic pressure, right ventricular hypertrophy, rise in oxidative stress, and increase in expression of α-SMA and TGF-β receptor 1 compared with MCT control group rats. In HUVECs, the expression of α-SMA was increased, whereas that of eNOS decreased after TGF-β exposure and was substantially reversed after pretreatment with benidipine. We concluded that benidipine and its combination with bosentan and sildenafil exhibit beneficial effects in MCT-induced PAH through the eNOS/TGF-β/α-SMA signaling pathway.

Abstract 2636: Epigenetic landscape of CD8+ T cell exhaustion and tissue residency in HPV-negative head and neck squamous cell carcinoma

Abstract Background Head and neck cancer is the 8th most common cancer worldwide. Human Papilloma Virus (HPV)-negative head and neck squamous cell carcinoma (HNSCC) overall survival rates remain low (25-40%) despite advances in treatments. CD8+ T cell differentiation into activated/cytotoxic (CTL), exhausted (TEX) and tissue-resident memory (TRM) phenotypes is a major determinant of successful cancer immunotherapy, which relies on the ability of CD8+ T cells to detect and eliminate cancer cells. Recent evidence suggests the importance of epigenetic regulation in the development of CD8+ T cell exhaustion and tissue residency; yet the epigenetic mechanisms that govern the transition between CTL, TEX, and TRM CD8+ T cell states remain unclear. This study investigates the epigenetic modifications associated with CTL, TEX, and TRM states using CD8+ T cells from healthy donors and HPV-negative HNSCC tumors. Methods An in vitro human CD8+ T cell stimulation assay using healthy donor cells was utilized to generate CTL, TEX and TRM phenotypes. Naïve T cells underwent CD3/28, TCR-independent stimulation for 9 days to promote CTL (3 days) and TEX (9 days) phenotypes, and TGF-β exposure (days 3-9) occurred to induce a TRM phenotype. Flow cytometry to confirm expression of cell-surface and intracellular markers for each phenotype is ongoing. Genome-wide mapping for activating (H3K4me1, H3K4me3) and repressive (H3K9me3, H3K27me3, H4K20me3) histone marks with CUT&Tag assays will be pursued for each of the in vitro CTL, TEX and TRM states, along with RNA-seq. CUT&Tag and RNA-seq will also characterize the epigenetic profiles of CD8+ T cells from single-cell suspensions generated from HPV-negative HNSCC tumors (n=5). Findings will be correlated with results from our in vitro T cell states. Results Preliminary flow cytometry confirmed increased expression of CD25, PD-1, Tim3 after 9 days of in vitro stimulation, supporting the induction of a CD8+ TEX phenotype. CD103 was also highly expressed in CD8+ T cells exposed to TGF-β, supporting the induction of the CD8+ TRM phenotype. Validation of the CTL state is ongoing. Phenotypic validation of cell states with flow cytometry (18 cell surface and intracellular markers) is ongoing. Feasibility of genome-wide mapping for activating and repressive histone marks with CUT&Tag on naïve CD8+ T cells has been confirmed. Conclusion This study investigates epigenetic changes that characterize the transition between CTL, TEX, and TRM states of CD8+ T cells using healthy donor cells through an in vitro assay and CD8+ T cells isolated from HPV-negative HNSCC tumors. Understanding the epigenetic profile of T cell states can help determine the major epigenetic regulators that lead to exhaustion and tissue residency, paving the way for improved T cell-based immunotherapies. Future experiments aim to validate our findings using TCR-specific in vitro and in vivo models. Citation Format: Marie K. Luff, Marco Craviero, Mohd Saleem Dar, Katherine McKinnon, Amir Kaskas, Clint T. Allen, Vassiliki Saloura. Epigenetic landscape of CD8+ T cell exhaustion and tissue residency in HPV-negative head and neck squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2636.

Caffeic acid ethanolamide induces antifibrosis, anti-inflammatory, and antioxidant effects protects against bleomycin-induced pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is a chronic and progressive interstitial lung disease; its cause is unknown, and it leads to notable health problems. Currently, only two drugs are recommended for IPF treatment. Although these drugs can mitigate lung function decline, neither can improve nor stabilize IPF or the symptoms perceived by patients. Therefore, the development of novel treatment options for pulmonary fibrosis is required. The present study investigated the effects of a novel compound, caffeic acid ethanolamide (CAEA), on human pulmonary fibroblasts and evaluated its potential to mitigate bleomycin-induced pulmonary fibrosis in mice. CAEA inhibited TGF-β-induced α-SMA and collagen expression in human pulmonary fibroblasts, indicating that CAEA prevents fibroblasts from differentiating into myofibroblasts following TGF-β exposure. In animal studies, CAEA treatment efficiently suppressed immune cell infiltration and the elevation of TNF-α and IL-6 in bronchoalveolar lavage fluid in mice with bleomycin-induced pulmonary fibrosis. Additionally, CAEA exerted antioxidant effects by recovering the enzymatic activities of oxidant scavengers. CAEA directly inhibited activation of TGF-β receptors and protected against bleomycin-induced pulmonary fibrosis through inhibition of the TGF-β/SMAD/CTGF signaling pathway. The protective effect of CAEA was comparable to that of pirfenidone, a clinically available drug. Our findings support the potential of CAEA as a viable method for preventing the progression of pulmonary fibrosis.

Molecular Regulation of Transforming Growth Factor-β1-induced Thioredoxin-interacting Protein Ubiquitination and Proteasomal Degradation in Lung Fibroblasts: Implication in Pulmonary Fibrosis.

Thioredoxin-interacting protein (TXNIP) plays a critical role in regulation of cellular redox reactions and inflammatory responses by interacting with thioredoxin (TRX) or the inflammasome. The role of TXNIP in lung fibrosis and molecular regulation of its stability have not been well studied. Therefore, here we investigated the molecular regulation of TXNIP stability and its role in TGF-β1-mediated signaling in lung fibroblasts. TXNIP protein levels were significantly decreased in lung tissues from bleomycin-challenged mice. Overexpression of TXNIP attenuated transforming growth factor-β1 (TGF-β1)-induced phosphorylation of Smad2/3 and fibronectin expression in lung fibroblasts, suggesting that decrease in TXNIP may contribute to the pathogenesis of lung fibrosis. Further, we observed that TGF-β1 lowered TXNIP protein levels, while TXNIP mRNA levels were unaltered by TGF-β1 exposure. TGF-β1 induced TXNIP degradation via the ubiquitin-proteasome system. A serine residue mutant (TNXIP-S308A) was resistant to TGF-β1-induced degradation. Furthermore, downregulationof ubiquitin-specific protease-13 (USP13) promoted the TGF-β1-induced TXNIP ubiquitination and degradation. Mechanistic studies revealed that USP13 targeted and deubiquitinated TXNIP. The results of this study revealed that the decrease of TXNIP in lungs apparently contributes to the pathogenesis of pulmonary fibrosis and that USP13 can target TXNP for deubiquitination and regulate its stability.

Upregulated LncRNA-Meg3 modulates the proliferation and survival of MEPM cells via interacting with Smad signaling in TCDD-induced cleft palate

Exposure to the environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in utero can result in high rates of cleft palate (CP) formation, yet the underlying mechanisms remain to be characterized. In vivo, the lncRNA Meg3 was upregulated following TCDD treatment in CP-associated murine embryonic palatal tissue, with concomitant changes in proliferative and apoptotic activity in these murine embryonic palatal mesenchymal (MEPM) cells. Meg3 can modulate the TGF-β/Smad to control the proliferation, survival, and differentiation of cells. Accordingly, TCCD and TGF-β1 were herein used to treat MEPM cells in vitro, revealing that while TCDD exposure altered the proliferative activity and apoptotic death of these cells, exogenous TGF-β1 exposure antagonized these effects via TGF-β/Smad signaling. TCDD promoted Meg3 upregulation, whereas TGF-β1 suppressed TCDD-driven upregulation of this lncRNA. Meg3 was additionally determined to directly interact with Smad2, with significant Meg3 enrichment in Smad2-immunoprecipitates following TCDD treatment. When Meg3 was silenced, the impact of TCDD on Smad signaling, proliferative activity, and apoptosis were ablated, while the effects of exogenous TGF-β1 were unchanged. This supports a model wherein Meg3 is upregulated in TCDD-exposed palatal tissue whereupon it can interact with Smad2 to suppress Smad-dependent signaling, thus controlling MEPM cell proliferation and apoptosis, contributing to TCDD-induced CP, which provides a theoretical support for the precautions of cleft palate induced by TCDD.

168 Impact of TGF-β exposure on IL-6 secretion in cystic fibrosis airway epithelia

168 Impact of TGF-β exposure on IL-6 secretion in cystic fibrosis airway epithelia

TGFβ1-RCN3-TGFBR1 loop facilitates pulmonary fibrosis by orchestrating fibroblast activation

BackgroundIdiopathic pulmonary fibrosis (IPF) bears high mortality due to unclear pathogenesis and limited therapeutic options. Therefore, identifying novel regulators is required to develop alternative therapeutic strategies.MethodsThe lung fibroblasts from IPF patients and Reticulocalbin 3 (RCN3) fibroblast-selective knockdown mouse model were used to determine the importance of Rcn3 in IPF; the epigenetic analysis and protein interaction assays, including BioID, were used for mechanistic studies.ResultsReticulocalbin 3 (RCN3) upregulation is associated with the fibrotic activation of lung fibroblasts from IPF patients and Rcn3 overexpression blunts the antifibrotic effects of pirfenidone and nintedanib. Moreover, repressing Rcn3 expression in mouse fibroblasts ameliorates bleomycin-induced lung fibrosis and pulmonary dysfunction in vivo. Mechanistically, RCN3 promotes fibroblast activation by maintaining persistent activation of TGFβ1 signalling via the TGFβ1-RCN3-TGFBR1 positive feedback loop, in which RCN3 upregulated by TGFβ1 exposure detains EZH2 (an epigenetic methyltransferase) in the cytoplasm through RCN3-EZH2 interaction, leading to the release of the EZH2-H3K27me3 epigenetic repression of TGFBR1 and the persistent expression of TGFBR1.ConclusionsThese findings introduce a novel regulating mechanism of TGFβ1 signalling in fibroblasts and uncover a critical role of the RCN3-mediated loop in lung fibrosis. RCN3 upregulation may cause resistance to IPF treatment and targeting RCN3 could be a novel approach to ameliorate pulmonary fibrosis.Graphical

Mechanical microscopy of cancer cells: TGF-β induced epithelial to mesenchymal transition corresponds to low intracellular viscosity in cancer cells.

Viscosity is an essential parameter that regulates bio-molecular reaction rates of diffusion-driven cellular processes. Hence, abnormal viscosity levels are often associated with various diseases and malfunctions like cancer. For this reason, monitoring intracellular viscosity becomes vital. While several approaches have been developed for in vitro and in vivo measurement of viscosity, analysis of intracellular viscosity in live cells has not yet been well realized. Our research introduces a novel, natural frequency-based, non-invasive method to determine the intracellular viscosity in cells. This method can not only efficiently analyze the differences in intracellular viscosity post modulation with molecules like PEG or glucose but is sensitive enough to distinguish the difference in intra-cellular viscosity among various cancer cell lines such as Huh-7, MCF-7, and MDAMB-231. Interestingly, TGF-β a cytokine reported to induce epithelial to mesenchymal transition (EMT), a feature associated with cancer invasiveness resulted in reduced viscosity of cancer cells, as captured through our method. To corroborate our findings with existing methods of analysis, we analyzed intra-cellular viscosity with a previously described viscosity-sensitive molecular rotor-based fluorophore-TPSII. In parity with our position sensing device (PSD)-based approach, an increase in fluorescence intensity was observed with viscosity enhancers, while, TGF-β exposure resulted in its reduction in the cells studied. This is the first study of its kind that attempts to characterize differences in intracellular viscosity using a novel, non-invasive PSD-based method.

Hybrid Material Based on Vaccinium myrtillus L. Extract and Gold Nanoparticles Reduces Oxidative Stress and Inflammation in Hepatic Stellate Cells Exposed to TGF-β.

(1) Background: The study aimed to investigate the impact of gold nanoparticles capped with Cornus sanguinea (NPCS) and mixed with a fruit extract (Vaccinum myrtillus L.-VL) on human hepatic stellate cells (LX-2) exposed to TGF-β. (2) Methods: NPCS were characterized by UV-Vis, transmission electron microscopy (TEM), zeta potential measurement, X-ray diffraction (XRD) and energy dispersive spectroscopy (EDX). The cytotoxic effects of VL, NPCS and of the hybrid compounds obtained by mixing the two components in variable proportions (NPCS-VL) were assessed. LDH activity, MDA levels, secretion of inflammation markers, the expression of fibrogenesis markers and collagen I synthesis were estimated after treating the cells with a mixture of 25:25 μg/mL NPCS and VL. (3) Results: TEM analysis showed that NPCS had spherical morphology and homogenous distribution, while their formation and elemental composition were confirmed by XRD and EDX analysis. TGF-β increased cell membrane damage as well as secretion of IL-1β, IL-1α and TLR4. It also amplified the expression of α-SMA and type III collagen and induced collagen I deposition. NPCS administration reduced the inflammation caused by TGF-β and downregulated α-SMA expression. VL diminished LDH activity and the secretion of proinflammatory cytokines. The NPCS-VL mixture maintained IL-1β, IL-1α, TLR4 and LDH at low levels after TGF-β exposure, but it enhanced collagen III expression. (4) Conclusions: The mixture of NPCS and VL improved cell membrane damage and inflammation triggered by TGF-β and mitigated collagen I deposition, but it increased the expression of collagen III, suggestive of a fibrogenetic effect of the hybrid material.

CFTR dysfunction in smooth muscle drives TGFβ dependent airway hyperreactivity

BackgroundThe primary underlying defect in cystic fibrosis (CF) is disrupted ion transport in epithelia throughout the body. It is unclear if symptoms such as airway hyperreactivity (AHR) and increased airway smooth muscle (ASM) volume in people with CF are due to inherent abnormalities in smooth muscle or are secondary to epithelial dysfunction. Transforming Growth Factor beta 1 (TGFβ) is an established genetic modifier of CF lung disease and a known driver of abnormal ASM function. Prior studies have demonstrated that CF mice develop greater AHR, goblet cell hyperplasia, and ASM hypertrophy after pulmonary TGFβ exposure. However, the mechanism driving these abnormalities in CF lung disease, specifically the contribution of CFTR loss in ASM, was unknown.MethodsIn this study, mice with smooth muscle-specific loss of CFTR function (Cftrfl/fl; SM-Cre mice) were exposed to pulmonary TGFβ. The impact on lung pathology and physiology was investigated through examination of lung mechanics, Western blot analysis, and pulmonary histology.ResultsCftrfl/fl; SM-Cre mice treated with TGFβ demonstrated greater methacholine-induced AHR than control mice. However, Cftrfl/fl; SM-Cre mice did not develop increased inflammation, ASM area, or goblet cell hyperplasia relative to controls following TGFβ exposure.ConclusionsThese results demonstrate a direct smooth muscle contribution to CF airway obstruction mediated by TGFβ. Dysfunction in non-epithelial tissues should be considered in the development of CF therapeutics, including potential genetic therapies.

Differentiation of IL-26+ TH17 intermediates into IL-17A producers via epithelial crosstalk in psoriasis

Interleukin (IL)-26 is a TH17 cytokine with known antimicrobial and pro-inflammatory functions. However, the precise role of IL-26 in the context of pathogenic TH17 responses is unknown. Here we identify a population of blood TH17 intermediates that produce high levels of IL-26 and differentiate into IL-17A-producing TH17 cells upon TGF-β1 exposure. By combining single cell RNA sequencing, TCR sequencing and spatial transcriptomics we show that this process occurs in psoriatic skin. In fact, IL-26+ TH17 intermediates infiltrating psoriatic skin induce TGF-β1 expression in basal keratinocytes and thereby promote their own differentiation into IL-17A-producing cells. Thus, our study identifies IL-26-producing cells as an early differentiation stage of TH17 cells that infiltrates psoriatic skin and controls its own maturation into IL17A-producing TH17 cells, via epithelial crosstalk involving paracrine production of TGF-β1.