Endocardial HDAC3 is required for myocardial trabeculation.

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

Spatiotemporal Evolution of Developing Palate in Mice.

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

Salivary transforming growth factor beta in oral submucous fibrosis: A diagnostic and predictive marker.

CONTEXT: Growth factors and cytokines like transforming growth factor beta (TGF-ß) play a key role in the pathogenesis of oral submucous fibrosis. AIMS: To elucidate the role of Salivary TGF-ß isoforms as a predictive and diagnostic marker for oral submucous fibrosis. SETTINGS AND DESIGN: A total of 30 OSMF and 10 control patients were included in this study, and their clinic-epidemiological data was recorded. METHODOLOGY: The expression of TGF-ß genes-TGF-ß1, TGF-ß2, TGF-ß3-was studied by a real-time polymerase chain reaction in tissue and saliva. Patients were given medicinal intervention for 12 weeks along with jaw-opening exercises. Expression of salivary TGF-ß genes was studied at 12 weeks. STATISTICAL ANALYSIS USED: SPSS software version 20. RESULT: Expression of salivary TGF beta isoforms in OSMF was more than in the control group. There was an increase in salivary TGF-ß1, ß2, ß3 expressions with increasing clinical grades of OSMF and advancing the stage of the disease. Expression of all the TGF beta isoforms was decreased after treatment with statistically significant results. Statistically significant correlations were found between the mean difference of TGF-ß1 and the mean difference between mouth opening and tongue protrusion. CONCLUSION: Salivary TGF-ß isoforms may be used in diagnosis, risk assessment, and screening of the entire population at risk of OSMF after its clinical validation. However, adequate sample size and segmental assessment of the expression of TGF-ß isoforms are needed for further evaluation.

Redifferentiation of genetically modified dedifferentiated chondrocytes in a microcavitary hydrogel.

OBJECTIVES: We genetically modified dedifferentiated chondrocytes (DCs) using lentiviral vectors and adenoviral vectors encoding TGF-ß3 (referred to as transgenic groups below) and encapsulated these DCs in the microcavitary hydrogel and investigated the combinational effect on redifferentiation of the genetically manipulated DCs. RESULTS: The Cell Counting Kit-8 data indicated that both transgenic groups exhibited significantly higher cell viability in the first week but inferior cell viability in the subsequent timepoints compared with those of the control group. Real-time polymerase chain reaction and western blot analysis results demonstrated that both transgenic groups had a better effect on redifferentiation to some extent, as evidenced by higher expression levels of chondrogenic genes, suggesting the validity of combination with transgenic DCs and the microcavitary hydrogel on redifferentiation. Although transgenic DCs with adenoviral vectors presented a superior extent of redifferentiation, they also expressed greater levels of the hypertrophic gene type X collagen. It is still worth further exploring how to deliver TGF-ß3 more efficiently and optimizing the appropriate parameters, including concentration and duration. CONCLUSIONS: The results demonstrated the better redifferentiation effect of DCs with the combinational use of transgenic TGF-ß3 and a microcavitary alginate hydrogel and implied that DCs would be alternative seed cells for cartilage tissue engineering due to their easily achieved sufficient cell amounts through multiple passages and great potential to redifferentiate to produce cartilaginous extracellular matrix.

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

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

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

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

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

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

CAV1 and KRT5 are potential targets for prostate cancer.

Prostate cancer is the most common malignant tumor of male urogenital system that occurs in prostate epithelium. However, relationship between CAV1 and KRT5 and prostate cancer remains unclear. The prostate cancer datasets GSE114740 and GSE200879 were downloaded from Gene Expression Omnibus generated by GPL11154 and GPL32170. De-batch processing was performed, differentially expressed genes (DEGs) were screened, and weighted gene co-expression network analysis. The construction and analysis of protein-protein interaction network, functional enrichment analysis, gene set enrichment analysis. Gene expression heat map was drawn and immune infiltration analysis was performed. Comparative toxicogenomics database analysis were performed to find the disease most related to core gene. In addition, the cell experiment was performed to verify the role of CAV1 and KRT5 by western blot. Divided into 4 groups: control, prostate cancer, prostate cancer-over expression, and prostate cancer- knock out. TargetScan screened miRNAs that regulated central DEGs; 770 DEGs were identified. According to Gene Ontology analysis, they were mainly concentrated in actin binding and G protein coupled receptor binding. In Kyoto Encyclopedia of Gene and Genome analysis, they were mainly concentrated in PI3K-Akt signal pathway, MAPK signal pathway, and ErbB signal pathway. The intersection of enrichment terms of differentially expressed genes and GOKEGG enrichment terms was mainly concentrated in ErbB signaling pathway and MAPK signaling pathway. Three important modules were generated. The protein-protein interaction network obtained 8 core genes (CAV1, BDNF, TGFB3, FGFR1, PRKCA, DLG4, SNAI2, KRT5). Heat map of gene expression showed that core genes (CAV1, TGFB3, FGFR1, SNAI2, KRT5) are highly expressed in prostate cancer tissues and low in normal tissues. Comparative toxicogenomics database analysis showed that core genes (CAV1, TGFB3, FGFR1, SNAI2, KRT5) were associated with prostate tumor, cancer, tumor metastasis, necrosis, and inflammation. CAV1 and KRT5 are up-regulated in prostate cancer. CAV1 and KRT5 are highly expressed in prostate cancer. The higher expression of CAV1 and KRT5, the worse prognosis.

Multi-ancestry epigenome-wide analyses identify methylated sites associated with aortic augmentation index in TOPMed MESA.

Despite the prognostic value of arterial stiffness (AS) and pulsatile hemodynamics (PH) for cardiovascular morbidity and mortality, epigenetic modifications that contribute to AS/PH remain unknown. To gain a better understanding of the link between epigenetics (DNA methylation) and AS/PH, we examined the relationship of eight measures of AS/PH with CpG sites and co-methylated regions using multi-ancestry participants from Trans-Omics for Precision Medicine (TOPMed) Multi-Ethnic Study of Atherosclerosis (MESA) with sample sizes ranging from 438 to 874. Epigenome-wide association analysis identified one genome-wide significant CpG (cg20711926-CYP1B1) associated with aortic augmentation index (AIx). Follow-up analyses, including gene set enrichment analysis, expression quantitative trait methylation analysis, and functional enrichment analysis on differentially methylated positions and regions, further prioritized three CpGs and their annotated genes (cg23800023-ETS1, cg08426368-TGFB3, and cg17350632-HLA-DPB1) for AIx. Among these, ETS1 and TGFB3 have been previously prioritized as candidate genes. Furthermore, both ETS1 and HLA-DPB1 have significant tissue correlations between Whole Blood and Aorta in GTEx, which suggests ETS1 and HLA-DPB1 could be potential biomarkers in understanding pathophysiology of AS/PH. Overall, our findings support the possible role of epigenetic regulation via DNA methylation of specific genes associated with AIx as well as identifying potential targets for regulation of AS/PH.

TGFB3 gene mutation associated with mandibular coronoid process hyperplasia: a family investigation.

OBJECTIVE: Coronoid process hyperplasia (CPH) of the mandible can lead to restricted mouth opening and maxillofacial deformities, which have been hypothesized to be closely associated with genetics. This study investigated the relationship between congenital CPH and TGFB3 mutation in a family of patients with CPH. STUDY DESIGN: A limited mouth opening proband with CPH underwent whole-exome gene sequencing in November 2019, and the results confirmed compound heterozygous mutations in the TGFB3 gene. Subsequently, clinical imaging and genetic testing were performed on 10 other individuals in his family. RESULTS: A total of 9 people in this family have CPH. Among them, 6 have the same exon compound heterozygous mutation sites of the TGFB3 gene (chr14-76446905 and chr14-76429713), accompanied by homozygous or heterozygous mutations in the 3'untranslated region (3'UTR) of the TGFB3 gene (chr14:76429555). The other 3 individuals have a homozygous mutation in the 3'untranslated region of the TGFB3 gene. CONCLUSION: The heterogeneous compound mutation of the TGFB3 gene or the homozygous mutation of 3'UTR of the TGFB3 gene may be correlated with CPH. In addition, the specifically related mechanism needs to be confirmed by further genetic animal experiments.

Transforming growth factor ß (TGF-ß) pathway in the immunopathogenesis of multiple sclerosis (MS); molecular approaches.

INTRODUCTION: Multiple sclerosis (MS) is an acute demyelinating disease with an autoimmune nature, followed by gradual neurodegeneration and enervating scar formation. Dysregulated immune response is a crucial dilemma contributing to the pathogenesis of MS. The role of chemokines and cytokines, such as transforming growth factor-ß (TGF-ß), have been recently highlighted regarding their altered expressions in MS. TGF-ß has three isoforms, TGF-ß1, TGF-ß2, and TGF-ß3, that are structurally similar; however, they can show different functions. RESULTS: All three isoforms are known to induce immune tolerance by modifying Foxp3+ regulatory T cells. Nevertheless, there are controversial reports concerning the role of TGF-ß1 and 2 in the progression of scar formation in MS. At the same time, these proteins also improve oligodendrocyte differentiation and have shown neuroprotective behavior, two cellular processes that suppress the pathogenesis of MS. TGF-ß3 shares the same properties but is less likely contributes to scar formation, and its direct role in MS remains elusive. DISCUSSION: To develop novel neuroimmunological treatment strategies for MS, the optimal strategy could be the one that causes immune modulation, induces neurogenesis, stimulates remyelination, and prevents excessive scar formation. Therefore, regarding its immunological properties, TGF-ß could be an appropriate candidate; however, contradictory results of previous studies have questioned its role and therapeutic potential in MS. In this review article, we provide an overview of the role of TGF-ß in immunopathogenesis of MS, related clinical and animal studies, and the treatment potential of TGF-ß in MS, emphasizing the role of different TGF-ß isoforms.

Let-7a-3p Inhibits Chondrogenic Differentiation of Bone Marrow Mesenchymal Stem Cells by Targeting TGF-ß3.

OBJECTIVE: Chondrogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) plays an important role in osteoarthritis (OA) treatment. Studies have reported the association of transforming growth factor beta-3 (TGF-ß3) with chondrogenic differentiation of BMSCs. In this study, the upstream mechanism and functions of TGF-ß3 in chondrogenic differentiation of BMSCs were explored. METHODS AND RESULTS: Flow cytometry was performed for the positive and negative MSC markers. Chondrogenic differentiation of BMSCs was evaluated by Alcian blue staining. Gene expression and protein expression levels were measured by RT-qPCR and western blotting, respectively. Relationship between let-7a-3p and TGF-ß3 was confirmed using bioinformatics analysis, luciferase reporter and RNA pulldown assays. Subcellular distributions of TGF-ß3 and let-7a-3p were determined by FISH. In this study, BMSCs were identified to possess the characteristics of mesenchymal stem cells. TGF-ß3 was found to induce chondrogenic differentiation of BMSCs. Mechanically, TGF-ß3 was verified to be targeted by let-7a-3p. In rescue assays, let-7a-3p overexpression reversed the effects of TGF-ß3 overexpression on chondrogenic differentiation of BMSCs. CONCLUSION: Let-7a-3p inhibits chondrogenic differentiation of bone marrow mesenchymal stem cells by targeting TGF-ß3.

Expression deregulation of matrix metalloproteinases and vasoconstriction related genes in Pakistani females with abnormal uterine bleeding.

BACKGROUND: Abnormal uterine bleeding (AUB) is irregular menstrual bleeding which has great impact on female health and life style. Various genetic factors are involved in etiology and pathology of AUB. Present study was designed to explore the association of PTGFR, MMP9, MMP2, TGFB3 and VEGFB with AUB. METHODS: Blood samples of 212 females with AUB were collected along with age-matched healthy control. Expression variation of targeted genes was evaluated using qPCR. Present study cohort was divided into different groups based on demographic parameters and all targeted genes were correlated with study demographics. RESULTS: Expression of targeted genes was significantly (P < 0.001) downregulated in females with AUB compared to control. Reduced (P < 0.01) expression of targeted genes was observed in all age groups (21-30, 31-40, 41-50 year) of AUB patients compared to respective control. Expression of VEGFB increased (P < 0.05) in AUB females with > 9 days bleeding compared to AUB patient had < 9 days bleeding. AUB women with miscarriage history showed upregulation in MMP2, TGFB3 (P < 0.05), and downregulation in MMP9 and VEGFB (P < 0.05) expression compared to AUB group with no miscarriage history. Expression of MMP2 increased (P < 0.05) in AUB females with > 60 kg body weigh compared to AUB patient with < 60 kg weight. CONCLUSION: Present study open a new window for diagnosis of AUB at early stages and suggested a possible involvement of PTGFR, MMP9, MMP2, TGFB3 and VEGFB as candidate biomarkers in AUB.

Dedifferentiated Schwann cell-derived TGF-ß3 is essential for the neural system to promote wound healing.

Rationale: Wound healing is among the most complicated physiological processes and requires the synchronization of various cell types with distinct roles to re-establish the condition of the original skin. Patients affected by peripheral neuropathies often experience failure to heal. Loss of Schwann cells (SCs), a crucial population of peripheral nervous system cells in skin, may contribute to chronic wounds. However, the role of SCs in wound healing are poorly understood. Methods: The activity of SCs was investigated by using a cell atlas of the wound healing process, which was generated by integrating single-cell RNA sequencing (scRNA-seq) libraries covering different states of mouse back skin. The results of in silico analysis were validated by in vitro cell culture and in vivo mouse model. Selective inhibitors and conditional RNAi by virus transfection were utilized to investigate the role of SCs in wound healing. Findings from mouse experiments were further verified in scRNA-seq analysis of diabetic patients. Results: Our in silico analysis revealed the heterogeneous cellular components of skin and the dynamic interactions of neural crest derived cells (NCs) with other cell types. We found that SCs dedifferentiated at an early stage of wound repair with upregulated Wnt signaling. We also identified dedifferentiated SC (dSC) defect in diabetic wounds in both mouse and human. Wnt inhibition at the wound site repressed SC dedifferentiation, leading to defective repair. Furthermore, dSCs derived TGF-ß3, which is context-dependent, promoted the migration of fibroblasts and keratinocytes. Moreover, TGF-ß3 supplementation enhanced the healing of chronic wounds in diabetic mice with impaired SCs. Conclusion: Our study thus advances the understanding of the roles of neural-derived cells in skin regeneration and suggests a potential therapeutic strategy for wound healing disorders.

Cytokine TGFß Gene Polymorphism in Asthma: TGF-Related SNP Analysis Enhances the Prediction of Disease Diagnosis (A Case-Control Study With Multivariable Data-Mining Model Development).

Introduction: TGF-ß and its receptors play a crucial role in asthma pathogenesis and bronchial remodeling in the course of the disease. TGF-ß1, TGF-ß2, and TGF-ß3 isoforms are responsible for chronic inflammation, bronchial hyperreactivity, myofibroblast activation, fibrosis, bronchial remodeling, and change the expression of approximately 1000 genes in asthma. TGF-ß SNPs are associated with the elevated plasma level of TGF-ß1, an increased level of total IgE, and an increased risk of remodeling of bronchi. Methods: The analysis of selected TGF-ß1, TGF-ß2, TGF-ß3-related single-nucleotide polymorphisms (SNP) was conducted on 652 DNA samples with an application of the MassARRAY® using the mass spectrometry (MALDI-TOF MS). Dataset was randomly split into training (80%) and validation sets (20%). For both asthma diagnosis and severity prediction, the C5.0 modelling with hyperparameter optimization was conducted on: clinical and SNP data (Clinical+TGF), only clinical (OnlyClinical) and minimum redundancy feature selection set (MRMR). Area under ROC (AUCROC) curves were compared using DeLong's test. Results: Minor allele carriers (MACs) in SNP rs2009112 [OR=1.85 (95%CI:1.11-3.1), p=0.016], rs2796821 [OR=1.72 (95%CI:1.1-2.69), p=0.017] and rs2796822 [OR=1.71 (95%CI:1.07-2.71), p=0.022] demonstrated an increased odds of severe asthma. Clinical+TGF model presented better diagnostic potential than OnlyClinical model in both training (p=0.0009) and validation (AUCROC=0.87 vs. 0.80,p=0.0052). At the same time, the MRMR model was not worse than the Clinical+TGF model (p=0.3607 on the training set, p=0.1590 on the validation set), while it was better in comparison with the Only Clinical model (p=0.0010 on the training set, p=0.0235 on validation set, AUCROC=0.85 vs. 0.87). On validation set Clinical+TGF model allowed for asthma diagnosis prediction with 88.4% sensitivity and 73.8% specificity. Discussion: Derived predictive models suggest the analysis of selected SNPs in TGF-ß genes in combination with clinical factors could predict asthma diagnosis with high sensitivity and specificity, however, the benefit of SNP analysis in severity prediction was not shown.

A mediator complex subunit 12 gain-of-function mutation induces partial leiomyoma cell properties in human uterine smooth muscle cells.

OBJECTIVE: To clarify whether a mediator complex subunit 12 (MED12) gain-of-function mutation induces leiomyoma cell properties in human uterine smooth muscle cells (USMCs). DESIGN: Experimental study. SETTING: Academic research laboratory. PATIENT(S): Women undergoing hysterectomy for leiomyoma. INTERVENTION(S): CRISPR/Cas9-mediated genome editing to introduce an MED12 gain-of-function mutation (G44D) into human USMCs. MAIN OUTCOME MEASURE(S): Cell proliferation, collagen production, and in vivo tumorigenicity of USMCs with vs. without the MED12 mutation. RESULT(S): Uterine smooth muscle cells isolated from the uterine myometrium of a 44-year-old patient were subjected to lentiviral vector-mediated gene transduction of the fluorescent protein Venus, followed by long-term passage. Uterine smooth muscle cells with a normal female karyotype, high cell proliferative activity, and Venus expression, but without stem/progenitor cell populations, were obtained and designated as USMC44. Using CRISPR/Cas9-mediated genome editing, mtUSMC44 (MED12, 131G>A, p.G44D) and mock USMC44 without MED12 mutation (wtUSMC44) were established from USMC44. wtUSMC44 and mtUSMC44 showed similar cell proliferation activity, even in the presence of estradiol and progesterone (EP) together with transforming growth factor-beta 3 (TGFB3). In addition, wtUSMC44 and mtUSMC44 generated similar tiny smooth muscle-like tissue constructs when xenotransplanted beneath the kidney capsule in immunodeficient mice treated with EP alone or TGFB3. In contrast, mtUSMC44 produced more collagen type I than wtUSMC in vitro, and this production was likely enhanced by EP and TGFB3. CONCLUSION(S): The results suggest that the MED12 gain-of-function mutation is involved in collagen production. Although approximately 70% of leiomyomas have MED12 mutations, additional factors and/or events other than MED12 and/or myometrial stem/progenitor cells may be required for fully inducing leiomyoma cell properties, including transformation, in USMCs.

Effects of Placenta-Derived Human Amniotic Epithelial Cells on the Wound Healing Process and TGF-ß Induced Scar Formation in Murine Ischemic-Reperfusion Injury Model.

BACKGROUND: Pressure ulcers (PUs), a result of ischemic reperfusion (IR) injuries, are prevalent skin problems which show refractoriness against standard therapeutic approaches. Besides, scar formation is a critical complication of ulcers that affects functionality and the skin's cosmetic aspect. The current study aimed to investigate the effects of placenta-derived human amniotic epithelial cells (hAECs), as important agents of regenerative medicine and stem cell therapy, on accelerating the healing of IR ulcers in mice. We also evaluated the effects of these cells on reducing the TGFß-induced scar formation. METHODS: Male Balb/c mice at the age of 6-8 weeks were subjected to three IR cycles. Afterward, the mice were divided into three experimental groups (n = 6 per group), including the control group, vehicle group, and hAECs treatment group. Mice of the treatment group received 100 µL of fresh hAECs 1 × 106 cell/ml suspension in PBS. Afterward, mice were assessed by histological, stereological, molecular, and western blotting techniques at 3, 7, 14, and 21 days after wounding. RESULTS: The histological and stereological results showed the most diminutive scar formation and better healing in the hAECs treated group compared to control group. Furthermore, our results demonstrated that the expression level of Col1A1 on days 3, 14, and 21 in the hAECs treated group was significantly lower than control. Additionally, injection of hAECs significantly reduced the expression level of Col3A1 on days 3, 7, and 21 while increased Col3A1 on the day 14. Otherwise, in the hAECs treated group, the expression levels of VEGFA on days 7 and 14 were higher, which showed that hAECs could promote angiogenesis and wound healing. Also, cell therapy significantly lowered the protein levels of TGF-ß1 on day 14, while the protein level of TGF-ß3 on day 14 was significantly higher. This data could demonstrate the role of hAECs in scar reduction in IR wounds. CONCLUSION: These results suggest that hAECs can promote re-epithelialization and wound closure in an animal model of PU. They also reduced scar formation during wound healing by reducing the expression of TGF-ß1/ TGF-ß3 ratio.

Maternal Perinatal Exposure to Dibutyl Phthalate Promotes Ovarian Dysfunction in Adult Female Offspring via Downregulation of TGF-ß2 and TGF-ß3.

Maternal exposure to dibutyl phthalate (DBP) may result in ovarian dysfunction in female offspring. However, the underlying mechanisms remain elusive. Pregnant Sprague-Dawley rats were intraperitoneally injected with different doses of DBP, estradiol, and corn oil from gestational day 7 until the end of lactation. The reproductive characteristics, mRNA, and protein expression of ovaries for the adult female offspring were compared. KGN cells were cultured in vitro with DBP, estrogen receptor antagonist, or ALK-5 inhibitor. Genes, proteins, estradiol, and progesterone expressed by KGN, cell proliferation, and apoptosis were measured respectively. Maternal perinatal exposure to DBP induced prolonged estrous period, increased secondary follicles, significant decreased mRNA, and protein levels of TGF-ß2, TGF-ß3, and TGF-ßRII in ovaries of the adult female offspring, but none difference for serum levels of sex hormones, ovarian TGF-ß1, and estrogen receptor. The mRNA levels of LHR, FSHR, and CYP19a in ovaries were also decreased. DBP might decrease the mRNA of TGF-ß2, TGF-ß3, and TGF-ßR II of KGN. DBP can inhibit the mRNA of CYP19 at 24 h, which might be blocked by the estrogen receptor antagonist, whose effects were attenuated at 48 h. DBP combined with FSH might time-dependently regulate the gene expression of TGF-ßR II, inhibitory at 24 h, but stimulative at 48 h, which could be blocked by the ALK5 inhibitor. However, the protein expressed by KGN was not influenced by DBP. DBP stimulated the proliferation of KGN at 24 h, which could be blocked by estrogen receptor antagonist, but attenuated at 48 h. The progesterone in culture medium secreted by KGN was decreased by DBP at 24 h. Maternal perinatal exposure to DBP induced decreased gene expression of TGF-ß signaling and functional proteins in ovaries of the adult female offspring. Molecular cross-talk between estrogen receptor and TGF-ß signaling pathway may play role in the mechanism of granulosa dysfunction induced by DBP.

Effect of passage number of genetically modified TGF-ß3 expressing primary chondrocytes on the chondrogenesis of ATDC5 cells in a 3D coculture system.

Due to the lack of blood vessels, nerves and lymphatics, articular cartilage is difficult to repair once damaged. Tissue engineering is considered to be a potential strategy for cartilage regeneration. Successful tissue engineering strategies depend on the effective combination of biomaterials, seed cells and biological factors. In our previous study, a genetically modified coculture system with chondrocytes and ATDC5 cells in an alginate hydrogel has exhibited a superior ability to enhance chondrogenesis. In this study, we further evaluated the influence of chondrocytes at various passages on chondrogenesis in the coculture system. The results demonstrated that transfection efficiency was hardly influenced by the passage number of chondrocytes. The coculture system with passage 5 (P5) chondrocytes had a better effect on chondrogenesis of ATDC5 cells, while chondrocytes in this coculture system presented higher levels of dedifferentiation than other groups with P1 or P3 chondrocytes. Therefore, P5 chondrocytes were shown to be more suitable for the coculture system, as they accumulated in sufficient cell numbers with more passages and had a higher level of dedifferentiation, which was prone to form a favorable niche for chondrogenesis of ATDC5 cells. This study may provide fresh insights for future cartilage tissue engineering strategies with a combination of a coculture system and advanced biomaterials.

Circ_0022382 ameliorated intervertebral disc degeneration by regulating TGF-ß3 expression through sponge adsorption of miR-4726-5p.

Circular RNAs (circRNAs) participate in the progression of many diseases, but knowledge on the role of circRNAs in intervertebral disc degeneration (IDD) is limited. In this study, we discovered the characteristics of a new circRNA (circ_0022382) in human endplate chondrocytes. Currently, real-time quantitative polymerase chain reaction (RT-qPCR) showed that the relative expression level of circ_0022382 was significantly lower under intermittent cyclic tension stimulation than in the control group. circ_0022382, miR-4726-5p and Transforming growth factor 3 (TGF-ß3) were evaluated by RT-qPCR, Western Blot and immunofluorescence assay. Additionally, the role and mechanism of circ_0022382 in vivo were also consistent in the rat model. Furthermore, Intermittent cyclic mechanical tension can cause degeneration of endplate chondrocytes. The tension-sensitive circRNA_0022382 was decreased, and we found that circRNA_0022382 promoted morphology of endplate chondrocytes by sponge-binding miR-4726-5p down-regulation of target gene the TGF-ß3 expression, thereby alleviating IDD. In a rat model of acupuncture, intervertebral disc injection of circ_0022382 relieved the progression of IDD in vivo. In conclusion, the circ_0022382/miR-4726-5p/TGF-ß3 axis plays a key role in the anabolism and catabolism of the endplate chondrocyte extracellular matrix (ECM). It is suggested that circ_0022382 may provide a new approach for the prevention and treatment of IDD.