Panax notoginseng Saponin Promotes Bone Regeneration in Distraction Osteogenesis via the TGF-β1 Signaling Pathway

Distraction osteogenesis (DO) is an efficient strategy that is employed for the treatment of large bone defects in craniomaxillofacial surgery. Despite its utility, however, DO is associated with a prolonged consolidation phase and a high complication rate that hinder its more widespread utilization. Panax notoginseng saponin (PNS) is a traditional Chinese medicine that is frequently administered for the treatment of a range of conditions. Herein, we explored the ability of PNS treatment to influence osteogenic differentiation using both rabbit bone marrow mesenchymal cells (BMSCs) and a model of mandibular DO. BMSC proliferation was assessed via CCK-8 assay, while osteogenic differentiation was monitored through ALP and alizarin red S staining. A PCR approach was used to evaluate the expression of genes associated with osteogenesis (ALP, Runx2, and OCN) and genes linked to the TGF pathway (TβR-II, SMAD2, SMAD3, and PPM1A). For in vivo experiments, treated BMSCs were locally injected into the DO gap, with PNS being injected into treated rabbits every other day throughout the experimental period. The quality of the regenerative process was assessed via scanning electron microscopy (SEM), energy dispersive spectroscopy (EDS), X-ray imaging, and hematoxylin and eosin (H&E) staining. These analyses revealed that PNS was able to promote BMSC osteogenesis and mandibular generation, driving the upregulation of osteogenesis-related genes at the mRNA levels through the modulation of the TGF-β1/Smad pathway. Consistently, the overexpression or silencing of TβR-II in PNS-treated BMSCs was sufficient to modulate their osteogenic potential. Analyses of in vivo mandibular DO outcomes revealed significantly augmented new bone growth in the PNS-treated group relative to control animals, with maximal osteogenesis in the group overexpressing rabbit TβR-II. Together, these results highlight the PNS as a promising and cost-effective therapeutic tool with the potential to enhance bone regeneration in clinical contexts through the modulation of the TGF-β1/Smad pathway.

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TRIM16 Positively Regulates Osteogenic Differentiation of Human Periodontal Ligament Stem Cells by Modulating the Ubiquitination and Degradation of RUNX2

10.21203/rs.3.rs-38207/v1 ◽

2020 ◽

Author(s):

Yi Zhao ◽

Qiaoli Zhai ◽

Hong Liu ◽

Xun Xi ◽

Shuai Chen ◽

...

Keyword(s):

Stem Cells ◽

Alkaline Phosphatase ◽

Bone Regeneration ◽

Osteogenic Differentiation ◽

Periodontal Ligament ◽

Osteogenic Potential ◽

Common Disease ◽

Periodontal Ligament Stem Cells ◽

Alizarin Red ◽

Periodontal Tissues

Abstract BackgroundPeriodontal disease is a common disease that compromises the integrity of tooth-supporting tissues. Bone regeneration is the ultimate goal of periodontal therapies, in which osteogenic differentiation of human periodontal ligament stem cells plays a critical role. The tripartite motif (TRIM)16 is downregulated in periodontal tissues of patients with periodontitis and involved in osteogenic differentiation of human bone marrow mesenchymal stem cells(hBMSCs).However, the role of TRIM16 in the osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs) is largely unknown.MethodshPDLSCs were isolated and identified by immunophenotype assays using flow cytometry. Overexpression plasmids and specific short-hairpin RNAs (shRNAs) were constructed to manipulate the expression of target molecules. Alkaline phosphatase (ALP) staining, alizarin red staining (ARS) and enzyme‐linked immunosorbent assays (ELISA) were used to evaluate osteogenic potential capacity. Reverse transcription quantitative PCR (RT-qPCR) and Western blot analysis were performed to determine the expression of osteogenic-related markers and activation of relevant signaling pathways. Co-immunoprecipitation assays were performed to confirm the interactions between proteins and the ubiquitination of RUNX2. A LC-MS/MS analysis was performed to explore the different expression proteins in present of TRIM16.ResultsTRIM16 significantly promoted alkaline phosphatase activity and mineralized nodule formation, and positively regulated the osteogenic differentiation of hPDLSCs by enhancing protein expression of RUNX2, COL1A1 and OCN. Mechanistically, TRIM16 serves as a pivotal factor that stabilizes RUNX2 protein levels by decreasing CHIP-mediated K48-linked ubiquitination degradation of the RUNX2 protein. Besides, TRIM16 significantly increased expression of COL1A1 via activation of p38MAPK/RUNX2.ConclusionThis study identified a novel mechanism of TRIM16 in regulating stability of the RUNX2 protein, which may promote the osteogenic differentiation of hPDLSCs. TRIM16 may be a potential target of stem cell based-bone regeneration for periodontal therapies.

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Dentin-derived Inorganic Minerals Promote the Osteogenesis of Bone Marrow-derived Mesenchymal Stem Cells: Potential Applications for Bone Regeneration

10.21203/rs.3.rs-34650/v1 ◽

2020 ◽

Author(s):

Gang Lei ◽

Yanqiu Wang ◽

Yan Yu ◽

Zehan Li ◽

Jiamin Lu ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Mesenchymal Stem Cells ◽

Bone Formation ◽

Bone Regeneration ◽

Osteogenic Differentiation ◽

Mapk Signaling ◽

Control Group ◽

Alizarin Red

Abstract Background Oral and maxillofacial bone loss is highly prevalent among populations and nowadays increased attention has been focused on dentin derivatives as desirable graft materials for bone regeneration. In this study, dentin-derived inorganic minerals (DIM) were fabricated with a high-temperature calcination technique and the effects of DIM on the osteogenic differentiation of bone marrow-derived mesenchymal stem cells (BMMSCs) and the bone formation were elucidated.Methods The effects of DIM on BMMSCs proliferation, apoptosis capacity were evaluated by CCK-8, flow cytometry and EdU assays. Alkaline phosphatase (ALP) activity detection, ALP staining, alizarin red staining and osteogenic markers expression analysis were performed to investigate the influence of DIM on the osteogenic differentiation of BMMSCs, as well as the relevant signal mechanisms. The model of critical-sized defects in calvarium of rats was constructed for exploring the in vivo efficiency of DIM on bone regeneration.Results Cell viability assays indicated that DIM had no cytotoxicity. BMMSCs cultured with DIM presented a higher level of osteogenic differentiation ability than those in the control group. The activation in ERK and p38 signals was detected in DIM-treated BMMSCs, and both pathways and osteogenic process were suppressed while using ERK inhibitor U0126 and p38 inhibitor SB203580, respectively. Furthermore, the animal experiments revealed that DIM could dramatically enhance new bone formation compared to the control group.Conclusion All these results demonstrated that DIM could promote BMMSCs osteogenic differentiation via triggering ERK and p38 MAPK signaling pathways and be a novel predictable material for facilitating bone formation.

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Dentin-Derived Inorganic Minerals Promote the Osteogenesis of Bone Marrow-Derived Mesenchymal Stem Cells: Potential Applications for Bone Regeneration

Stem Cells International ◽

10.1155/2020/8889731 ◽

2020 ◽

Vol 2020 ◽

pp. 1-16

Author(s):

Gang Lei ◽

Yanqiu Wang ◽

Yan Yu ◽

Zehan Li ◽

Jiamin Lu ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Mesenchymal Stem Cells ◽

Bone Formation ◽

Bone Regeneration ◽

Osteogenic Differentiation ◽

Mapk Signaling ◽

Control Group ◽

Alizarin Red

Background. Oral and maxillofacial bone loss is highly prevalent among populations, and nowadays, increased attention has been focused on dentin derivatives serving as desirable graft materials for bone regeneration. In this study, dentin-derived inorganic mineral (DIM) was fabricated with a high-temperature calcination technique and the effects of DIM on the osteogenic differentiation of bone marrow-derived mesenchymal stem cells (BMMSCs) and the bone formation were elucidated. Methods. The effects of DIM on BMMSC proliferation and apoptosis capacity were evaluated by CCK-8, flow cytometry, and EdU assays. Alkaline phosphatase (ALP) activity detection, ALP staining, alizarin red staining, and osteogenic marker expression analysis were performed to investigate the influence of DIM on the osteogenic differentiation of BMMSCs, as well as the relevant signal mechanisms. The model of critical-sized defects in the calvarium of rats was constructed for exploring the in vivo efficiency of DIM on bone regeneration. Results. Cell viability assays indicated that DIM had no cytotoxicity. BMMSCs cultured with DIM presented a higher level of osteogenic differentiation ability than those in the control group. The activation in ERK and p38 signals was detected in DIM-treated BMMSCs, and both pathways and osteogenic process were suppressed while using ERK inhibitor U0126 and p38 inhibitor SB203580, respectively. Furthermore, the animal experiments revealed that DIM could dramatically enhance new bone formation compared to the control group. Conclusion. DIM could promote BMMSC osteogenic differentiation via triggering the ERK and p38 MAPK signaling pathways and might be a novel predictable material for facilitating bone formation.

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Temporal induction of Lhx8 by optogenetic control system for efficient bone regeneration

Stem Cell Research & Therapy ◽

10.1186/s13287-021-02412-8 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Delan Huang ◽

Runze Li ◽

Jianhan Ren ◽

Haotian Luo ◽

Weicai Wang ◽

...

Keyword(s):

Alkaline Phosphatase ◽

Control System ◽

Bone Regeneration ◽

Osteogenic Differentiation ◽

Blue Light ◽

Calvarial Defect ◽

Defect Model ◽

Alizarin Red ◽

Optogenetic Control

Abstract Background The spatiotemporal regulation of essential genes is crucial for controlling the growth and differentiation of cells in a precise manner during regeneration. Recently, optogenetics was considered as a potent technology for sophisticated regulation of target genes, which might be a promising tool for regenerative medicine. In this study, we used an optogenetic control system to precisely regulate the expression of Lhx8 to promote efficient bone regeneration. Methods Quantitative real-time PCR and western blotting were used to detect the expression of Lhx8 and osteogenic marker genes. Alkaline phosphatase staining and alizarin red staining were used to detect alkaline phosphatase activity and calcium nodules. A customized optogenetic expression system was constructed to regulate Lhx8, of which the expression was activated in blue light but not in dark. We also used a critical calvarial defect model for the analysis of bone regeneration in vivo. Moreover, micro-computed tomography (micro-CT), three-dimensional reconstruction, quantitative bone measurement, and histological and immunohistochemistry analysis were performed to investigate the formation of new bone in vivo. Results During the osteogenic differentiation of BMSCs, the expression levels of Lhx8 increased initially but then decreased thereafter. Lhx8 promoted the early proliferation of BMSCs but inhibited subsequent osteogenic differentiation. The optogenetic activation of Lhx8 in BMSCs in the early stages of differentiation by blue light stimulation led to a significant increase in cell proliferation, thus allowing a sufficient number of differentiating BMSCs to enter the later osteogenic differentiation stage. Analysis of the critical calvarial defect model revealed that the pulsed optogenetic activation of Lhx8 in transplanted BMSCs over a 5-day period led to a significant increase in the generation of bone in vivo. Conclusions Lhx8 plays a critical role in balancing proliferation and osteogenic differentiation in BMSCs. The optogenetic activation of Lhx8 expression at early stage of BMSCs differentiation led to better osteogenesis, which would be a promising strategy for precise bone regeneration.

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Role of Fzd6 in Regulating the Osteogenic Differentiation of Adipose-derived Stem Cells in Osteoporosis

10.21203/rs.3.rs-328417/v1 ◽

2021 ◽

Author(s):

Tianli Wu ◽

Zhihao Yao ◽

Gang Tao ◽

Fangzhi Lou ◽

Hui Tang ◽

...

Keyword(s):

Stem Cells ◽

Osteogenic Differentiation ◽

Wnt Signaling Pathway ◽

Osteogenic Potential ◽

Adipose Derived Stem Cells ◽

Alizarin Red ◽

Differentiation Potential

Abstract Objective: Although it has been demonstrated that adipose-derived stem cells (ASCs) from osteoporosis mice (OP-ASCs) exhibit impaired osteogenic differentiation potential, the molecular mechanism has not yet been elucidated. We found that Fzd6 was decreased in OP-ASCs compared with ASCs. This study investigates the effects and underlying mechanisms of Fzd6 in the osteogenic potential of OP-ASCs. Methods: Fzd6 expression in ASCs and OP-ASCs was measured by PCR gene chip. Fzd6 overexpression and silencing lentiviruses were used to evaluate the role of Fzd6 in the osteogenic differentiation of OP-ASCs. Real-time PCR (qPCR) and western blotting (WB) was performed to detect the expression of Fzd6 and bone-related molecules, including runt-related transcription factor 2 (Runx2) and osteopontin (Opn). Alizarin red staining and Alkaline phosphatase (ALP) staining was performed following osteogenic induction. Microscopic CT (Micro-CT), hematoxylin and eosin staining (H&E) staining, and Masson staining were used to assess the role of Fzd6 in osteogenic differentiation of osteoporosis (OP) mice in vivo.Results: Expression of Fzd6 was decreased significantly in OP-ASCs. Fzd6 silencing down-regulated the osteogenic ability of OP-ASCs in vitro. Overexpression of Fzd6 rescued the impaired osteogenic capacity in OP-ASCs in vitro. We obtained similar results in vivo.Conclusions: Fzd6 plays an important role in regulating the osteogenic ability of OP-ASCs both in vivo and in vitro. Overexpression of Fzd6 associated with the Wnt signaling pathway promotes the osteogenic ability of OP-ASCs, which provides new insights for the prevention and treatment of OP.

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EGFL6 regulates angiogenesis and osteogenesis in distraction osteogenesis via Wnt/β-catenin signaling

Stem Cell Research & Therapy ◽

10.1186/s13287-021-02487-3 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Junjie Shen ◽

Yi Sun ◽

Xuanzhe Liu ◽

Yu Zhu ◽

Bingbo Bao ◽

...

Keyword(s):

Bone Formation ◽

Osteogenic Differentiation ◽

Distraction Osteogenesis ◽

Bone Repair ◽

Angiogenic Factor ◽

Osteogenic Potential ◽

Human Umbilical Cord ◽

Alizarin Red ◽

Differentiation Potential

Abstract Background Osteogenesis is tightly coupled with angiogenesis during bone repair and regeneration. However, the underlying mechanisms linking these processes remain largely undefined. The present study aimed to test the hypothesis that epidermal growth factor-like domain-containing protein 6 (EGFL6), an angiogenic factor, also functions in bone marrow mesenchymal stem cells (BMSCs), playing a key role in the interaction between osteogenesis and angiogenesis. Methods We evaluated how EGFL6 affects angiogenic activity of human umbilical cord vein endothelial cells (HUVECs) via proliferation, transwell migration, wound healing, and tube-formation assays. Alkaline phosphatase (ALP) and Alizarin Red S (AR-S) were used to assay the osteogenic potential of BMSCs. qRT-PCR, western blotting, and immunocytochemistry were used to evaluate angio- and osteo-specific markers and pathway-related genes and proteins. In order to determine how EGFL6 affects angiogenesis and osteogenesis in vivo, EGFL6 was injected into fracture gaps in a rat tibia distraction osteogenesis (DO) model. Radiography, histology, and histomorphometry were used to quantitatively evaluate angiogenesis and osteogenesis. Results EGFL6 stimulated both angiogenesis and osteogenic differentiation through Wnt/β-catenin signaling in vitro. Administration of EGFL6 in the rat DO model promoted CD31hiEMCNhi type H-positive capillary formation associated with enhanced bone formation. Type H vessels were the referred subtype involved during DO stimulated by EGFL6. Conclusion EGFL6 enhanced the osteogenic differentiation potential of BMSCs and accelerated bone regeneration by stimulating angiogenesis. Thus, increasing EGFL6 secretion appeared to underpin the therapeutic benefit by promoting angiogenesis-coupled bone formation. These results imply that boosting local concentrations of EGFL6 may represent a new strategy for the treatment of compromised fracture healing and bone defect restoration.

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EGFL6 Regulates the Interaction Between Angiogenesis and Osteogenesis via Wnt/β-catenin Signaling in Distraction Osteogenesis

10.21203/rs.3.rs-251914/v1 ◽

2021 ◽

Author(s):

Junjie Shen ◽

Yi Sun ◽

Xuanzhe Liu ◽

Yu Zhu ◽

Bingbo Bao ◽

...

Keyword(s):

Bone Formation ◽

Osteogenic Differentiation ◽

Distraction Osteogenesis ◽

Bone Repair ◽

Angiogenic Factor ◽

Osteogenic Potential ◽

Human Umbilical Cord ◽

Alizarin Red ◽

Differentiation Potential

Abstract Background: Osteogenesis is tightly coupled with angiogenesis during bone repair and regeneration. However, the underlying mechanisms linking these processes remain largely undefined. The present study aimed to test the hypothesis that epidermal growth factor-like domain-containing protein 6 (EGFL6), an angiogenic factor, also functions in bone marrow mesenchymal stem cells (BMSCs) and plays a key role in the interaction between osteogenesis angiogenesis.Methods: We evaluated how EGFL6 affects angiogenic activity of human umbilical cord vein endothelial cells (HUVECs) via proliferation, transwell migration, wound healing, and tube-formation assays. Alkaline phosphatase (ALP) and Alizarin Red S (AR-S) were used to assay the osteogenic potential of BMSCs. qRT-PCR, western blotting, and immunocytochemistry were used to evaluate angio- and osteo-specific markers and pathway-related genes and proteins. In order to determine how EGFL6 affects angiogenesis and osteogenesis in vivo, EGFL6 was injected into fracture gaps in a rat tibia distraction osteogenesis (DO) model. Radiography, histology, and histomorphometry were used to quantitatively evaluate angiogenesis and osteogenesis. Results: EGFL6 stimulated both angiogenesis and osteogenic differentiation through Wnt/β-catenin signaling in vitro. Administration of EGFL6 in the rat DO model promoted CD31hiEMCNhi type H-positive capillary formation associated with enhanced bone formation. Type H vessels were the referred subtype involved during DO stimulated by EGFL6.Conclusion: EGFL6 enhanced osteogenic differentiation potential of BMSCs and accelerated bone regeneration by stimulating angiogenesis, thus exerting therapeutic benefit by increasing EGFL6 secretion to promote angiogenesis-coupled bone formation. These results imply that boosting local concentrations of EGFL6 may represent a new strategy for the treatment of compromised fracture healing and bone defect restoration.

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miR-129-5p Promotes Osteogenic Differentiation of BMSCs and Bone Regeneration via Repressing Dkk3

Stem Cells International ◽

10.1155/2021/7435605 ◽

2021 ◽

Vol 2021 ◽

pp. 1-18

Author(s):

Changming Zhao ◽

Yulin Gu ◽

Yan Wang ◽

Qiaozhen Qin ◽

Ting Wang ◽

...

Keyword(s):

Western Blot ◽

Bone Regeneration ◽

Osteogenic Differentiation ◽

Luciferase Reporter ◽

Luciferase Reporter Gene ◽

Alizarin Red ◽

Gene Assay

Objective. Accumulating evidence indicates that microRNAs (miRNAs) play crucial roles in osteogenic differentiation. However, the associated mechanisms remain elusive. This paper is aimed at exploring the role of miR-129-5p in regulating bone marrow mesenchymal stem cell (BMSC) differentiation and bone regeneration in vivo and in vitro. Methods. BMSCs were transduced by miR-129-5p mimic, miR-129-5p inhibitor, and negative control lentivirus. The ability of BMSC differentiation to osteoblast was tested by alkaline phosphatase (ALP) and alizarin red staining (ARS). The expression of osteogenic genes (Runx2, Bmp2, and OCN) was examined via quantitative RT-PCR and western blot. A mouse model of calvaria defect was investigated by Micro-CT, immunohistochemistry, and histological examination. The luciferase reporter gene assay was performed to confirm the binding between Dkk3 and miR-129-5p. For the transfection experiments, lipofectamine 3000 was used to transfect pcDNA-Dkk3 into BMSCs to overexpress Dkk3. Coimmunoprecipitation and immunofluorescent localization assay were included for exploring the role of Dkk3 and β-catenin. Results. miR-129-5p was induced in BMSCs and MSC cell line C3H10T1/2 cells under osteogenic medium. Overexpression of miR-129-5p significantly promoted osteogenic differentiation of BMSCs in vitro. Moreover, BMSCs transduced with miR-129-5p mimic exhibited better bone regeneration compared with BMSCs transduced with control counterpart in vivo. Luciferase and western blot data showed that Dickkopf3 (Dkk3) is a target gene of miR-129-5p and the expression of Dkk3 was inhibited in BMSCs transduced with miR-129-5p mimic but enhanced in BMSCs transduced with miR-129-5p inhibitor. In addition, Dkk3 interacted with β-catenin directly. Conclusions. miR-129-5p promotes osteogenic differentiation of BMSCs and bone regeneration, and miR-129-5p/Dkk3 axis may be new potential targets for the treatment of bone defect and bone loss.

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Epigenetic Mechanism of TGF-β1 Promoting Osteogenic Differentiation of Human Bone Marrow Mesenchymal Stem Cells in Osteoarthritis

10.21203/rs.3.rs-732375/v1 ◽

2021 ◽

Author(s):

Jianwei He ◽

Weiwei Cao ◽

Qinzheng Fang ◽

Inayat Azeem ◽

Wei Liu

Keyword(s):

Osteogenic Differentiation ◽

Bone Alkaline Phosphatase ◽

Epigenetic Mechanism ◽

Mrna Levels ◽

Osteogenic Medium ◽

Alizarin Red ◽

Expression Levels ◽

Qrt Pcr ◽

Tgf Β1

Abstract Objectives: It had been proved that TGF-β1 was correlated with onset of osteoarthritis in vitro and vivo. Here, This study was to elucidate the epigenetic mechanism of TGF-β1 promoting osteogenic differentiation in osteoarthritis. Methods: hBMSCs surface antigens were assayed by flow cytometry tests. qRT-PCR was performed to detect hBMSCs mRNA levels of RUNX2, PPARγ and SOX9. hBMSCs were stained by osteoalkaline phosphatase and alizarin red. The qRT-PCR and Western blot were both used to detect the expression levels of methylases, demethylases and osteogenic transcription factor RUNX2 after hBMSCs were cultrued in osteogenic medium coincubated with TGF-β1 solution. Results: hBMSCs were identified by over expressions of CD90, CD105 and CD44, as well as the positive multi-diffenentiation potential tests. hBMSCs bone alkaline phosphatase and alizarin red staining were observed to deepen in TGF-β1 group compared with the osteogenic culture group. The mRNA expression levels of EZH1, KDM2B, KDM4A/4B/4C/4D, and KDM6A /6B were increased in hBMSCs cultured in osteogenic medium. The expression levels of KDM6A/6B were shown increasement when TGF-β1 was co-incubated with osteogenic medium. Furthermore, the mRNA and protein levels of KDM6A/6B were significantly decreased after SB431542 was added in the medium. RUNX2 was significantly inhibited by the addition of GSK-J4 solution, while KDM6A/6B expression level did not change significantly. Conclusion: The osteogenic differentiation of hBMSCs was related to the enhanced expressions of EZH1, KDM2B, KDM4A-4D, KDM6A/6B. The expression levels of demethylase KDM6A/6B were positively regulated by the TGF-β/Smad signaling pathway, which promoted the osteogenic differentiation of hBMSCs.

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Concurrent YAP/TAZ and SMAD signaling mediate vocal fold fibrosis

Scientific Reports ◽

10.1038/s41598-021-92871-z ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Ryosuke Nakamura ◽

Nao Hiwatashi ◽

Renjie Bing ◽

Carina P. Doyle ◽

Ryan C. Branski

Keyword(s):

Vocal Fold ◽

Vocal Folds ◽

Nuclear Accumulation ◽

Smad Pathway ◽

Surgical Interventions ◽

Smad Signaling ◽

Iatrogenic Damage ◽

Tgf Β1

AbstractVocal fold (VF) fibrosis is a major cause of intractable voice-related disability and reduced quality of life. Excision of fibrotic regions is suboptimal and associated with scar recurrence and/or further iatrogenic damage. Non-surgical interventions are limited, putatively related to limited insight regarding biochemical events underlying fibrosis, and downstream, the lack of therapeutic targets. YAP/TAZ integrates diverse cell signaling events and interacts with signaling pathways related to fibrosis, including the TGF-β/SMAD pathway. We investigated the expression of YAP/TAZ following vocal fold injury in vivo as well as the effects of TGF-β1 on YAP/TAZ activity in human vocal fold fibroblasts, fibroblast-myofibroblast transition, and TGF-β/SMAD signaling. Iatrogenic injury increased nuclear localization of YAP and TAZ in fibrotic rat vocal folds. In vitro, TGF-β1 activated YAP and TAZ in human VF fibroblasts, and inhibition of YAP/TAZ reversed TGF-β1-stimulated fibroplastic gene upregulation. Additionally, TGF-β1 induced localization of YAP and TAZ in close proximity to SMAD2/3, and nuclear accumulation of SMAD2/3 was inhibited by a YAP/TAZ inhibitor. Collectively, YAP and TAZ were synergistically activated with the TGF-β/SMAD pathway, and likely essential for the fibroplastic phenotypic shift in VF fibroblasts. Based on these data, YAP/TAZ may evolve as an attractive therapeutic target for VF fibrosis.

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