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Variations in diuretic-mediated inhibition of carbonic anhydrase-dependent chloride transport in platelets and vascular smooth muscle could account for the contrasting efficacy of the thiazide and thiazide-like diuretics in reducing cardiovascular morbidity in patients with hypertension. We assessed platelet carbonic anhydrase activity and catecholamine-induced platelet aggregation in the presence of a thiazide and a "thiazide-like" inhibitor of the sodium-chloride cotransporter. Individual variation in platelet carbonic anhydrase activity correlated with contrasting sensitivity to epinephrine-mediated platelet aggregation. Both chlorthalidone, which potently inhibits platelet carbonic anhydrase, and bendroflumethiazide, which has much less effect on this enzyme, increased the amount of epinephrine needed to induce platelet aggregation when compared with the absence of a diuretic. However, chlorthalidone was significantly more effective than bendroflumethiazide in reducing epinephrine-mediated platelet aggregation. Chlorthalidone also induced marked changes in the number of gene transcripts for two proteins that mediate angiogenesis and vascular permeability, vascular endothelial growth factor C and transforming growth factor-beta3; chlorthalidone and bendroflumethiazide had contrasting effects on the expression of vascular endothelial growth factor C. Chlorthalidone and bendroflumethiazide reduced vascular permeability to albumin, but only chlorthalidone increased angiogenesis. Thiazides and thiazide-like diuretics can comparably reduce blood pressure, but the drugs in this class are not all alike. It can be suggested from our findings that thiazide and thiazide-like diuretics vary in their pleiotropic effects on platelets and in the vasculature, and these differences could explain the contrasting ability of these drugs to reduce cardiovascular morbidity despite comparable reduction in blood pressure. [ABSTRACT FROM AUTHOR]

Regarding cartilage repair, tissue engineering is currently focusing on the use of adult mesenchymal stem cells (MSC) as an alternative to autologous chondrocytes. The potential of stem cells from various tissues to differentiate towards the chondrogenic phenotype has been investigated and it appears that the most common and studied sources are bone marrow (BM) and adipose tissue (AT) for historical and easy access reasons. In addition to three dimensional environment, the presence of member(s) of the transforming growth factor (TGF-β family and low oxygen tension have been reported to promote the in vitro differentiation of MSCs. Our work aimed at characterizing and comparing the degree of chondrogenic differentiation of MSCs isolated from BM and AT cultured in the same conditions. We also further aimed at and at determining whether hypoxia (2% oxygen) could affect the chondrogenic potential of AT-MSCs. Cells were first expanded in the presence of FGF-2, then harvested and centrifuged to allow formation of cell pellets, which were cultured in the presence of TGF-β3 and/or Bone Morphogenetic Protein-2 (BMP-2) and with 2 or 20% oxygen tension, for 24 days. Markers of the chondrocyte (COL2A1, AGC1, Sox9) and hypertrophic chondrocyte (COL10A1, MMP-13) were monitored by real-time PCR and/or by immunohistological staining. Our data show that BMP-2/TGF-β3 combination is the best culture condition to induce the chondrocyte phenotype in pellet cultures of BM and AT-MSCs. Particularly, a switch in the expression of the pre-chondrogenic type IIA form to the cartilage-specific type IIB form of COL2A1 was observed. A parallel increase in gene expression of COL10A1 and MMP-13 was also recorded. However when AT-MSCs were cultured in hypoxia, the expression of markers of hypertrophic chondrocytes decreased when BMP-2/TGF-β3 were present in the medium. Thus it seems that hypoxia participates to the control of AT-MSCs chondrogenesis. Altogether, these cellular model systems will help us to investigate further the potential of different adult stem cells for cartilage engineering. [ABSTRACT FROM AUTHOR]

Abstract: Purpose: In the chick embryo, the administration of cadmium (Cd) induces ventral body wall defects (VBWDs) similar to the human omphalocele. Transforming growth factors β (TGFs-β) are involved in many developmental processes, including ventral body wall formation. The Tgfβ2−/− Tgfβ3−/− double knockout mice and Tgfβ2−/− Tgfβ3+/− mutants show VBWD, whereas Tgfβ2+/− Tgfβ3−/− mutants display normal ventral body wall fusion. We designed this study to investigate the messenger RNA (mRNA) levels of TGF-β2 and TGF-β3 in the Cd-induced omphalocele chick model during early embryogenesis. Methods: Chick embryos were exposed to either Cd or saline, harvested 1 hour (1H), 4H, and 8H after treatment and then divided into 2 groups: control and Cd (n = 8 at each time-point, respectively). Reverse transcription polymerase chain reaction was performed to evaluate the mRNA levels of TGF-β2 and TGF-β3 and statistically analyzed. Results: The mRNA expression levels of TGF-β2 at 1H were significantly decreased in the Cd group compared to controls (P < .05). However, the levels of TGF-β3 were not altered at all the time-points studied. Conclusion: We provide evidence, for the first time, that TGF-β2 gene expression is downregulated during a narrow window of early embryogenesis in the Cd chick model. Our data show that TGF-β2 is the key gene involved in the formation of ventral body wall. [Copyright &y& Elsevier]

Transforming growth factor-β (TGF-β) plays crucial roles in controlling cell differentiation and maintaining tissue integrity. Previously we reported that TGF-β3 treatment decreased the mRNA expression of the gap junction protein, connexin 43 as well as cell number, which lead to the inhibition of chondrogenic condensation in cultured chick leg bud mesenchymal cells. The present study demonstrates that TGF-β3 can induce cleavage in the ectodomain of neuronal cadherin (N-cadherin) at the initiation stage of chondrogenesis and reduce cell numbers, cellular adhesion and the expression level of connexin 43. Differential displayed PCR (DD-PCR) comparison of adherent- and non-adherent chick leg chondrogenic progenitor cells showed increased expression of the chick ras-responsive element binding transcription factor, cRREB-1, in adherent cells. In chick leg bud mesenchymal cells, cRREB-1 transcription was inhibited by TGF-β3 at the early stage of chondrogenesis. Small interfering RNA (siRNA)-mediated knockdown of cRREB-1 reduced cell numbers, cellular adhesion, and the expression level of connexin 43 resulting in the inhibition of precartilage condensation. Taken together, these findings indicate that TGF-β3 mediates the inhibitory signal necessary for precartilage condensation by stimulating N-cadherin shedding and reducing cRREB-1 expression levels. [ABSTRACT FROM AUTHOR]

Transforming growth factor-βs (TGF-βs), expressed in various tissues, play important roles in embryonic development and adult tissue homeostasis through their effects on cell proliferation, cell differentiation, cell death, and cell motility. However, expression of TGF-β signaling components and their biological effect on taste epithelia has not been elucidated. We performed expression analysis of TGF-β signaling components in taste epithelia and found that the TGF-β3 mRNA was specifically expressed in taste buds. Type II TGF-βs receptor (TβR-II) mRNA was specifically expressed in the tongue epithelia including the taste epithelia. To elucidate the biological function of TGF-β3 in taste epithelia, we performed proliferation assay with primary cultured taste epithelial cells. In the presence of TGF-β3, percentage of BrdU-labeled cells decreased significantly, suggesting that the TGF-β3 inhibited the proliferation of cultured taste epithelial cells through inhibiting cell-cycle entry into S phase. By quantitative reverse transcription–polymerase chain reaction assay, we found that the TGF-β3 resulted in an increased level of expression of p15Ink4b and p21Cip1, suggesting that the TGF-β3 inhibited the taste epithelial cell proliferation through inhibiting G1cyclin–Cdk complexes. Taken together, these results suggested that the TGF-β3 may regulate taste epithelial cell homeostasis through controlling cell proliferation. [ABSTRACT FROM AUTHOR]

Uterine leiomyoma are common, benign tumors that are enriched in extracellular matrix. The tumors are characterized by a disoriented and loosely packed collagen fibril structure similar to other diseases with disrupted Transforming growth factor β (TGF-β) signaling. Here we characterized TGF-β3 signaling and the expression patterns of the critical extracellular matrix component versican in leiomyoma and myometrial tissue and cell culture. We also demonstrate the regulation of the versican variants by TGF-β3. Using leiomyoma and matched myometrium from 15 patients, messenger RNA (mRNA) from leiomyoma and myometrium was analyzed by semiquantitative real time reverse transcription-polymerase chain reaction (RT-PCR), while protein analysis was done by western blot. Transforming growth factor β3 transcripts were increased 4-fold in leiomyoma versus matched myometrium. Phosphorylated-TGF-β RII and phosphorylated-Smad 2/3 complex were greater in leiomyoma as documented by Western blot. The inhibitor Smad7 transcripts were decreased 0.44-fold. The glycosaminoglycan (GAG)-rich versican variants were elevated in leiomyoma versus myometrial tissue: specifically V0 (4.27 ± 1.12) and V1 (2.01± 0.27). Treatment of leiomyoma and myometrial cells with TGF-β3 increased GAG-rich versican variant expression 7 to 12 fold. Neutralizing TGF-β3 antibody decreased the expression of the GAG-rich versican variants 2 to 8 fold in leiomyoma cells. Taken together, the aberrant production of excessive and disorganized extracellular matrix that defines the leiomyoma phenotype involves the activation of the TGF-β signaling pathway and excessive production of GAG-rich versican variants. [ABSTRACT FROM AUTHOR]

Abstract: During spermatogenesis in mammalian testes, junction restructuring takes place at the Sertoli–Sertoli and Sertoli–germ cell interface, which is coupled with germ cell development, such as cell cycle progression, and translocation of the germ cell within the seminiferous epithelium. In the rat testis, restructuring of the blood–testis barrier (BTB) formed between Sertoli cells near the basement membrane and disruption of the apical ectoplasmic specialization (apical ES) between Sertoli cells and fully developed spermatids (spermatozoa) at the luminal edge of the seminiferous epithelium occur concurrently at stage VIII of the seminiferous epithelial cycle of spermatogenesis. These two processes are essential for the translocation of primary spermatocytes from the basal to the apical compartment to prepare for meiosis, and the release of spermatozoa into the lumen of the seminiferous epithelium at spermiation, respectively. Cytokines, such as TNFα and TGFβ3, are present at high levels in the microenvironment of the epithelium at this stage of the epithelial cycle. Since these cytokines were shown to disrupt the BTB integrity and germ cell adhesion, it was proposed that some cytokines released from germ cells, particularly primary spermatocytes, and Sertoli cells, would induce restructuring of the BTB and apical ES at stage VIII of the seminiferous epithelial cycle. In this review, the intricate role of cytokines and testosterone to regulate the transit of primary spermatocytes at the BTB and spermiation will be discussed. Possible regulators that mediate cytokine-induced junction restructuring, including gap junction and extracellular matrix, and the role of testosterone on junction dynamics in the testis will also be discussed. [Copyright &y& Elsevier]

Abstract: Prenatal exposure to diazepam, a prototype sedative drug that belongs to Benzodiazepines, can lead to orofacial clefting in human newborns. By using real-time PCR, in the present study we investigated whether diazepam elicits gene expression alterations in extracellular matrix (ECM) components, growth factors and gamma-aminobutyric acid receptor (GABRB3), implicated in the coordinate regulation of palate development. Palate fibroblasts were treated with diazepam (Dz-N fibroblasts) and compared to cleft lip-palate (CLP) fibroblasts obtained from patients with no known exposure to diazepam or other teratogens. Untreated fibroblasts from non-CLP patients were used as control. The results showed significant convergences in gene expression pattern of collagens, fibromodulin, vitronectin, tenascin C, integrins and metalloprotease MMP13 between Dz-N and CLP fibroblasts. Among the growth factors, constitutive Fibroblast Growth Factor 2 (FGF2) was greatly enhanced in Dz-N and CLP fibroblasts and associated with a higher reduction of FGF receptor. Transforming Growth Factor beta 3 (TGFβ3) resulted up-regulated in CLP fibroblasts and decreased in Dz-N fibroblasts. We found phenotypic differences exhibited by Dz-N and CLP fibroblasts in GABRB3 gene regulation, so further studies are necessary to determine whether GABAergic system could be involved in the development of diazepam mediated CLP phenotype. Taken together the results elucidate the molecular mechanisms underlying possible toxicology effects induced by diazepam. Counselling of women on the safety of diazepam exposure is clinically important, also for the forensic consequences. [Copyright &y& Elsevier]

Mesenchymal stem cells (MSCs), especially genetically modified MSCs, have become a promising therapeutic approach for the treatment of rheumatoid arthritis (RA) through modulating immune responses. However, most MSCs used in the treatment of RA are modified based on a single gene. In this study, we evaluated the therapeutic effects of human BMSCs (hBMSCs) with COX-2 silence and TGF-β3 overexpression in the treatment of RA in a rabbit model. hBMSCs were cotransfected with shCOX-2 and TGF-β3 through lentiviral vector delivery. After SPIO-Molday ION Rhodamine-B™ (MIRB) labeling, lenti-shCOX2-TGF-β3 hBMSCs, lenti-shCOX2 hBMSCs, lenti-TGF-β3 hBMSCs, hBMSCs without genetic modification, or phosphate-buffered saline (PBS) were injected into the knee joint of rabbits with antigen-induced arthritis (AIA). The diameter of the knee joint and soft-tissue swelling score (STS) were recorded, and the levels of inflammatory mediators, including interleukin-1β (IL-1β), tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), and prostaglandin E2 (PGE2) were evaluated by ELISA. Clinical 3.0T MR imaging (MRI) was used to track the distribution and dynamic migration of hBMSCs in the joint. Histopathological and immunohistochemical assays were conducted to localize labeled hBMSCs and assess the alteration of synovial hyperplasia, inflammatory cell infiltration, and cartilage damage. COX-2 silencing and TGF-β3 overexpression in hBMSCs were confirmed through real-time PCR and Western blot analyses. Reduced joint diameter, soft-tissue swelling (STS) score, and PGE2, IL-1β, and TNF-α expression were detected 4 weeks after injection of MIRB-labeled lenti-shCOX2-TGF-β3 hBMSCs into the joint in rabbits with AIA. Eight weeks after hBMSC injection, reduced inflammatory cell infiltration, improved hyperplasia of the synovial lining, recovered cartilage damage, and increased matrix staining were observed in joints injected with lenti-shCOX2-TGF-β3 hBMSCs and lenti-shCOX2 hBMSCs. Slight synovial hyperplasia, no surface fibrillation, and strong positive expression of collagen II staining in chondrocytes and cartilage matrix were detected in the joints 12 weeks after injection of lenti-shCOX2-TGF-β3 hBMSCs. In addition, hBMSCs were detected by MRI imaging throughout the process of hBMSC treatment. Intra-articular injection of hBMSCs with COX-2 silence and TGFβ3 overexpression not only significantly inhibited joint inflammation and synovium hyperplasia, but also protected articular cartilage at the early stage. In addition, intra-articular injection of hBMSCs with COX-2 silence and TGFβ3 overexpression promoted chondrocyte and matrix proliferation. This study provides an alternative therapeutic strategy for the treatment of RA using genetically modified hBMSCs. [ABSTRACT FROM AUTHOR]

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-β3) 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-β3 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-β3 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. • Circ_0022382 promotes TGF-β3 expression. • Circ_0022382/miR-4726-5p/TGF-β3 axis • Circ_0022382 ameliorates IDD. [ABSTRACT FROM AUTHOR]

Abstract: The objective of this study was to investigate the influence of dynamic compressive loading on chondrogenesis of mesenchymal stem cells (MSCs) in the presence of TGF-β3. Isolated porcine MSCs were suspended in 2% agarose and subjected to intermittent dynamic compression (10% strain) for a period of 42 days in a dynamic compression bioreactor. After 42 days in culture, the free-swelling specimens exhibited more intense alcian blue staining for proteoglycans, while immunohistochemical analysis revealed increased collagen type II immunoreactivity. Glycosaminoglycan (GAG) content increased with time for both free-swelling and dynamically loaded constructs, and by day 42 it was significantly higher in both the core (2.5±0.21%w/w vs. 0.94±0.03%w/w) and annulus (1.09±0.09%w/w vs. 0.59±0.08%w/w) of free-swelling constructs compared to dynamically loaded constructs. This result suggests that further optimization is required in controlling the biomechanical and/or the biochemical environment if such stimuli are to have beneficial effects in generating functional cartilaginous tissue. [Copyright &y& Elsevier]

Abstract: The aim of this study was to create a gene expression profile to better define the phenotype of human adipose-derived stromal cells (HADSCs) during in vitro chondrogenesis, osteogenesis and adipogenesis. A novel aspect of this work was the analysis of the same subset of genes during HADSC differentiation into all three lineages. Chondrogenic induction resulted in increased mRNA expression of Sox transcription factors, COL2A1, COL10A1, Runx2, and Osterix. This is the first report demonstrating significant upregulation in expression of osteogenesis-related transcription factors Runx2 and Osterix by TGF-β3 induction of HADSCs during in vitro chondrogenesis. These findings suggest that the commonly-used chondrogenic induction reagents promote differentiation suggestive of hypertrophic chondrocytes and osteoblasts. We conclude that alternative strategies are required to induce efficient articular chondrocyte differentiation in order for HADSCs to be of clinical use in cartilage tissue engineering. [Copyright &y& Elsevier]

Although palatal shelf adhesion is a crucial event during palate development, little work has been carried out to determine which molecules are responsible for this process. Furthermore, whether altered palatal shelf adhesion causes the cleft palate presented by Tgf-β3 null mutant mice has not yet been clarified. Here, we study the presence/distribution of some extracellular matrix and cell adhesion molecules at the time of the contact of palatal shelves in both wild-type and Tgf-β3 null mutant palates of two strains of mice (C57/BL/6J (C57), and MF1) that develop cleft palates of different severity. We have performed immunohistochemistry with antibodies against collagens IV and IX, laminin, fibronectin, the α5- and β1-integrins, and ICAM-1; in situ hybridization with a Nectin-1 riboprobe; and palatal shelf cultures treated or untreated with TGF-β3 or neutralizing antibodies against fibronectin or the α5-integrin. Our results show the location of these molecules in the wild-type mouse medial edge epithelium (MEE) of both strains at the time of the contact of palatal shelves; the heavier (C57) and milder (MF1) alteration of their presence in the Tgf-β3 null mutants; the importance of TGF-β3 to restore their normal pattern of expression; and the crucial role of fibronectin and the α5-integrin in palatal shelf adhesion. We thus provide insight into the molecular bases of this important process and the cleft palate presented by Tgf-β3 null mutant mice. [ABSTRACT FROM AUTHOR]

Axonal regeneration at a site of peripheral nerve repair can be impeded by the formation of scar tissue, which creates a mechanical barrier and initiates the development of multiple branched axonal sprouts that form a neuroma. We have investigated the hypothesis that the application of a scar-reducing agent to the nerve repair site would permit better axonal regeneration. In anaesthetised C57 Black-6 mice, the left sciatic nerve was sectioned and immediately re-approximated using four epineurial sutures. In five groups of eight mice, we injected transforming growth factor-β3 (50 or 500 ng), interleukin-10 (IL-10) (125 or 500 ng), or saline into and around the repair site, both before and after the nerve section. Another group of eight animals acted as sham-operated controls. After 6 weeks, the outcome was assessed by recording compound action potentials (CAPs), measuring collagen levels using picrosirius red staining, and counting the number of myelinated axons proximal and distal to the repair. CAPs evoked by electrical stimulation distal to the repair were significantly smaller in all repair groups except for the low-dose IL-10 group, where they were not significantly different from that in controls. The area of staining for collagen had significantly increased in all repair groups except for the low-dose IL-10 group, which was not significantly different from that in controls. The myelinated fibre counts were always higher distal to the repair site, but there were no significant differences between groups. We conclude that administration of a low-dose of IL-10 to a site of sciatic nerve repair reduces scar formation and permits better regeneration of the damaged axons. [ABSTRACT FROM AUTHOR]

Summary: Objective: To determine whether the functional properties of tissue-engineered constructs cultured in a chemically-defined medium supplemented briefly with TGF-β3 can be enhanced with the application of dynamic deformational loading. Methods: Primary immature bovine cells (2–3 months old) were encapsulated in agarose hydrogel (2%, 30×106 cells/ml) and cultured in chemically-defined medium supplemented for the first 2 weeks with transforming growth factor beta 3 (TGF-β3) (10μg/ml). Physiologic deformational loading (1Hz, 3h/day, 10% unconfined deformation initially and tapering to 2% peak-to-peak deformation by day 42) was applied either concurrent with or after the period of TGF-β3 supplementation. Mechanical and biochemical properties were evaluated up to day 56. Results: Dynamic deformational loading applied concurrently with TGF-β3 supplementation yielded significantly lower (−90%) overall mechanical properties when compared to free-swelling controls. In contrast, the same loading protocol applied after the discontinuation of the growth factor resulted in significantly increased (+10%) overall mechanical properties relative to free-swelling controls. Equilibrium modulus values reach 1306±79kPa and glycosaminoglycan levels reach 8.7±1.6% w.w. during this 8-week period and are similar to host cartilage properties (994±280kPa, 6.3±0.9% w.w.). Conclusions: An optimal strategy for the functional tissue engineering of articular cartilage, particularly to accelerate construct development, may incorporate sequential application of different growth factors and applied deformational loading. [Copyright &y& Elsevier]

Abstract: Transforming growth factor-β2 and -β3 (TGF-β2 and -β3) are important members of TGF-β family which play important roles in the growth, maintenance, and repair processes of developing embryos, neonates, and adults. Preparation of large quantities of these two cytokines, which is necessary for structural studies and other applications, has proven to be extremely difficult. We have developed a novel Chinese hamster ovary cell-based expression system for high-level expression and high recovery of recombinant human TGF-β2 and -β3. In this system, we used a mammalian expression vector which contains a glutamine synthetase coding region for amplification, together with a modified TGF-β2 or -β3 open reading frame for expression. The leader peptide of TGF-β2 or -β3 was replaced by that from the V-J2-C region of a mouse immunoglobulin κ-chain, and a poly-histidine tag was inserted immediately after the leader sequence to facilitate protein purification without changing the mature TGF-β2 or -β3 amino acid sequence. In addition, the extreme N-terminal cysteine residue of TGF-β2 or -β3 was replaced by a serine residue. The resulting expression constructs produced two stable cell clones expressing 10mg of TGF-β2 and 8mg of TGF-β3 per liter of spent medium. The purification scheme involved the use of two simple chromatographic steps with a typical yield of 5mg of TGF-β2 and 4mg of TGF-β3. This method represents a significant improvement over previously published methods and may be applicable to other TGF-β superfamily members. We further confirmed that latent TGF-β2 and -β3 can be activated by proteolysis and glycolysis, which have not been reported before. [Copyright &y& Elsevier]

Abstract: We previously reported evidence that patients with periodontitis have serum antibodies to oral Gram positive bacteria that are cross-reactive with epithelial antigens, including CD24. High level expression of CD24 was confined to the reactive periodontal epithelium and inflamed gingival attachment. As a model for the reactive epithelium of chronic periodontitis, H413 epithelial cells derived from a human oral squamous cell carcinoma were cloned and lines expressing high levels of CD24 were selected. RNA interference protocols were designed to determine if CD24 could modulate intercellular interactions and regulate the biology of these epithelial cells. Knock-down of CD24 protein was demonstrated by Western blot and flow cytometry. The level of mRNA for CD24 was reduced 90% by RNAi treatment as assayed by real-time, reverse transcriptase (RT)-PCR. Gene products known to be important in epithelial biology, including E-cadherin and TGF-β3 that were demonstrated to undergo altered expression patterns in the periodontal lesion, were investigated. Down-regulation of CD24 mRNA was associated with reduced e-cadherin expression and up-regulated expression of snail, twist, and tgf-β3. The cells were treated with monoclonal antibodies to CD24 to mimic the action of auto-reactive antibodies to CD24 detected in affected patients. Relative to isotype control antibody, stimulation by anti-CD24 antibodies induced up-regulated expression of e-cadherin and down-regulation of tgf-β3 as assessed by real-time RT-PCR. No consistent changes for expression of β-catenin, connexins, integrins, icam-1, tgf-β1 or tgf-β2 were observed. CD24 could play an important role in modulating expression of genes that regulate epithelial differentiation in periodontal disease. [Copyright &y& Elsevier]

Abstract: Transforming growth factor-β3 (TGF-β3) has a specific role in vivo in the patterning of embryonic and tissue-specific gene expression. We have cloned and sequenced the mouse TGF-β3 5′-flanking region to study the transcriptional regulation of this gene. Promoter fragments were cloned into a promoterless luciferase reporter plasmid to study functional activity in a human skin melanoma cell line A375 (A375). Sequential 5′-deletion encompassing DNA sequences from −2297 to −1003bp exhibited high promoter activity in A375 cells, whereas the promoter activity decreased to minimal in the −742 to 104bp regions, suggesting both positive and negative transcriptional regulation in the TGF-β3 promoter. The fragment containing 1.8kb had the highest luciferase activity. Characterization of this 1.8kb 5′-flanking region upstream of the translation start site showed a putative hunchback-binding site consensus sequence. The electrophoretic mobility shift assay (EMSA) and transient transfection experiments showed that the putative hunchback-binding site is functional and regulated TGF-β3 promoter transcriptional activity. The DNA-complex including the hunchback sequence binding protein (HbSBP) was important for suppression of the promoter activity in A375 cells. Mutation of the hunchback consensus sequence resulted in up to 2-fold higher promoter activity than the wild type construct. There was an absence of HbSBP in other cell lines tested including 3T3 fibroblast and B-16 mouse skin melanoma as determined by EMSA and Western blot analysis. HbSBP may function as a TGF-β3 gene transcriptional regulator and may be expressed in a cell type-specific manner. [Copyright &y& Elsevier]

LIM-homeodomain (Lhx) genes constitute a gene family that plays critical roles in the control of pattern formation and cell type specification. We have identified a chicken L3/Lhx8 gene, which was widely expressed in the craniofacial region. Whole-mount in situ hybridization showed that L3/Lhx8 mRNA was expressed from stage 15--31 HH in overlapping domains of the maxillary process. Frozen sections revealed these signals in the mesenchyme underneath the epithelium. To determine whether the expression of L3/Lhx8 in the maxillary primordia required signals from the overlying oral epithelium, maxillary processes of stage 23 HH chick embryos were transplanted into the limb bud, in which the mesenchyme was grown in the presence or absence of oral epithelium. The maxillary mesenchyme with epithelium showed significant levels of L3/Lhx8 gene expression. In contrast, no expression of L3/Lhx8 was detected in the epithelium-free mesenchyme. To further explore signaling molecule(s) responsible for Lhx induction, a bead, soaked in either Fgf-8b or TGF-β3, was implanted into an epithelium-free mesenchymal graft. Both TGF-β3 and Fgf-8b beads induced expressions of L3/Lhx8 in epithelium-free mesenchymal grafts. Our data suggest that the L3/Lhx8 gene contributes to epithelial mesenchymal interaction in facial morphogenesis and that Fgf-8b and TGF-β3 were, at least in part, responsible for the Lhx expression in the maxillary process. [ABSTRACT FROM AUTHOR]

Objective: To evaluate the clinical features and the expression of transforming growth factor-β3 (TGF-β3) and connective tissue growth factor (CTGF) in myometrium and uterine leiomyomas after preoperative treatment with gonadotropin-releasing hormone-analogs (GnRH-a) and tibolone. Methods: Twenty-three patients received 3.75 mg leuprolide acetate depot for 4 months. Twenty-two patients received the same therapy plus 2.5 mg tibolone daily. Patients underwent uterine surgery after therapy. Twenty-two untreated patients underwent surgery directly. Hematologic tests, bone mineral density (BMD) measurement, and ultrasonographic evaluation of uterine volume were performed before and after treatment. Menorrhagia and pelvic pain were evaluated with a visual analog scale. Hot flushes were recorded in daily diaries. Immunohistochemical expression of TGF-β3 and CTGF in myometrium and myoma samples was evaluated semiquantitatively. Results: After therapy, hemoglobin and iron levels similarly increased in both groups. BMD significantly decreased only in the GnRH-a group. Uterine volume similarly decreased in both groups. No patient had menorrhagia or pelvic pain at the end of therapy. The number of hot flushes increased after the first month in the GnRH-a group; in the GnRH-a plus tibolone group, it remained constant and was lower. In untreated cases, TGF-β3 and CTGF smooth muscle cell immunoexpression was lower in myometrium than in leiomyomas. After medical treatment, growth factor immunoexpression remained unchanged in myometrial samples and was reduced in leiomyomas. Endothelial cells showed strong immunopositivity, both in untreated and in treated cases. Conclusion: This study focuses on the effects of GnRH-a and tibolone on TGF-β3 and CTGF expression in myometrium and myomas and supports the hypothesis of a pathogenetic role of these growth factors in uterine fibromatosis. [Copyright &y& Elsevier]

Summary: Objective: The use of cytokines as localized therapeutic agents is limited by the lack of a satisfactory delivery system. The aim of the current investigation was to determine the release kinetics and bioactivity of a simplified cytokine/collagen gel system designed to achieve extended, local delivery of bioactive cytokines at sites of premature cranial suture fusion (craniosynostosis). Design: Cytokine release was determined by ELISA measurements of Tgf-β3 collected in media. Cytokine bioactivity was determined by measuring the effect of conditioned media, containing released Tgf-β3, on mink lung epithelial cell proliferation and osteoblast alkaline phosphatase activity. Osteoblast response was evaluated by measuring proliferation of cells cultured on collagen gel containing Tgf-β3 using an AlamarBlue assay. Results: Gels loaded with 100 and 500ng of Tgf-β3 produced a sustained release over 14 days with a pattern of initial large release followed by a gradual reduction in the amount released over the time. The reduced release over time was correlated to the amount initially loaded. Mink lung epithelial cell assay results indicated that Tgf-β3 released from the collagen gel retained its bioactivity following incorporation into the collagen gel and release into the media. This bioactivity was further illustrated by a decreased alkaline phosphatase activity measured in osteoblasts cultured on the gels loaded with Tgf-β3. Osteoblast proliferation assays demonstrated that the collagen gel has an inherent inhibitory effect on osteoblast cell number. Conclusions: This collagen gel/cytokine delivery system can retain and release bioactive cytokine over a prolonged period. These results will allow for better optimization of future in vitro and in vivo studies directed at improving the treatment of craniosynostosis. [Copyright &y& Elsevier]

Abstract: Chondrogenesis is a critical step in palatogenesis. All-trans retinoic acid (atRA), a vitamin A derivative, is a known teratogenic effector of cleft palate. Here, we evaluated the effects of atRA on the osteo-/chondrogenic differentiation of mouse embryonic palate mesenchymal (MEPM) cells. MEPM cells, in a high-density micromass environment, undergo active chondrogenesis in a manner analogous to that of limb-derived mesenchymal cells, and served as a valid model system to investigate the mechanisms regulating chondrogenesis during palatogenesis. atRA-treated MEPM micromass expressed relatively higher levels of osteoblastic gene markers (alkaline phosphatase and collagen type I) and lower levels of chondrocytic gene markers (collagen type II and aggrecan). As transforming growth factor-β3 (TGF-β3) is an essential growth factor for chondrogenesis of embryonic mesenchymal cells both in in vivo and in vitro conditions, we thereby explored the effects of atRA on TGF-β3 signaling pathway. atRA led to an increase in mRNA expression of TGF-β3 and an instantaneous decrease in TGF-β type II receptor (TβRII) as determined by real-time RT-PCR. Further study showed that atRA inhibited phosphorylation of Smad2 and Smad3 and increased Smad7 expression. Activation of the Smad pathways by transfection with Smad7ΔC mutant or constitutively active TβRII retroviral vector abolished atRA-induced inhibition of chondrogenesis as indicated by Alcian blue staining, indicating that Smad signaling is essential for this response. Taken together, these data for the first time demonstrated a role for RA-induced hypochondrogenesis through regulation of the TGF-β3 pathway and suggested a role for TβRII /Smad in retinoid-induced cleft palate. [Copyright &y& Elsevier]

Abstract: In this study we investigated whether signalling by TGF-β3 and Wnt-5a cross-talk during chondrogenic differentiation of chick wing mesenchyme. Using differential display polymerase chain reaction screening, we found the expression of Wnt-5a to be significantly increased during transforming growth factor-β3 (TGF-β3)-induced precartilage condensation in mesenchyme micromass cultures. Transfection of cells with a Wnt-5a expression construct promoted precartilage condensation and chondrogenesis in micromass cultures, similar to that observed when chondrogenic-competent cells were exposed to TGF-β3. Overexpression of Wnt-5a or treatment with TGF-β3 stimulated the activation of protein kinase C-α (PKC-α) and p38 mitogen-activated protein kinase (MAPK), both positive regulators of chondrogenic differentiation. Inactivation of PKC-α and p38 MAPK by specific inhibitors abrogated chondrogenesis stimulated by both TGF-β3 and Wnt-5a. Similarly, partial reduction in TGF-β3-induced Wnt-5a expression by small interfering RNA resulted in decreased activities of PKC-α and p38 MAPK, and abolished the chondro-stimulatory effect of TGF-β3. Collectively, these findings indicate that Wnt-5a, a non-canonical Wnt, can mediate the chondro-stimulatory effect of TGF-β3 through upregulation of PKC-α and p38MAPK signaling. [Copyright &y& Elsevier]

Abstract: In the mammalian testis, preleptotene and leptotene spermatocytes residing in the basal compartment of the seminiferous epithelium must traverse the blood-testis barrier (BTB) at late stage VIII through early stage IX of the epithelial cycle during spermatogenesis, entering the adluminal compartment for further development. However, until recently the regulatory mechanisms that regulate BTB dynamics remained largely unknown. We provide a critical review regarding the significance of cytokines in regulating the ‘opening’ and ‘closing’ of the BTB. We also discuss how cytokines may be working in concert with adaptors that selectively govern the downstream signaling pathways. This process, in turn, regulates the dynamics of either Sertoli–Sertoli tight junction (TJ), Sertoli–germ cell adherens junction (AJ), or both junction types in the epithelium, thereby permitting TJ opening without compromising AJs, and vice versa. We also discuss how adaptors alter their protein–protein association with the integral membrane proteins at the cell–cell interface via changes in their phosphorylation status, thereby altering adhesion function at AJ. These findings illustrate that the testis is a novel in vivo model to study the biology of junction restructuring. Furthermore, a molecular model is presented regarding how cytokines selectively regulate TJ/AJ restructuring in the epithelium during spermatogenesis. [Copyright &y& Elsevier]

Several members of the transforming growth factor (TGF)-β superfamily are expressed in developing teeth from the initiation stage through adulthood. Of those, TGF-β1 regulates odontoblast differentiation and dentin extracellular matrix synthesis. However, the molecular mechanism of TGF-β3 in dental pulp cells is not clearly understood. In the present study, beads soaked with human recombinant TGF-β3 induced ectopic mineralization in dental pulp from fetal mouse tooth germ samples, which increased in a dose-dependent manner. Further, TGF-β3 promoted mRNA expression, and increased protein levels of osteocalcin (OCN) and type I collagen (COL I) in dental pulp cells. We also observed that the expression of dentin sialophosphoprotein and dentin matrix protein 1 was induced by TGF-β3 in primary cultured dental pulp cells, however, not in calvaria osteoblasts, whereas OCN, osteopontin and osteonectin expression was increased after treatment with TGF-β3 in both dental pulp cells and calvaria osteoblasts. Dentin sialoprotein was also partially detected in the vicinity of TGF-β3 soaked beadsin vivo. These results indicate for the first time that TGF-β3 induces ectopic mineralization through upregulation of OCN and COL I expression in dental pulp cells, and may regulate the differentiation of dental pulp stem cells to odontoblasts. [ABSTRACT FROM AUTHOR]

P311 is an 8-kDa protein originally found in neurons and muscle. We recently showed that expression of P311 in NIH 3T3 cells induced a myofibroblast phenotype with low TGF-β1 expression. Here we demonstrate that P311 downregulates not only TGF-β1, but also TGF-β2, expression, with no effect on TGF-β3. In addition, P311 interacts with TGF-β2 in a yeast two-hybrid system through a sequence encompassing part of the TGF-β latent associated protein (LAP) and part of mature TGF-β2. Coimmunoprecipitations demonstrated interaction between P311 and TGF-β1 and 2, but not TGF-β3. Additional coimmunoprecipitations after introducing LAP or mature TGF-β1 into cells demonstrated P311 binding to LAP, but not to mature TGF-β. P311 has a conserved PEST domain, which generally serves as a rapid degradation signal. Deletion of the PEST domain reversed the effect of P311 on TGF-β isoforms. Finally, Smad3 activity was decreased in P311-expressing cells, but was corrected by exogenous TGF-β1 treatment, which also elevated TGF-β1 mRNA level. This suggested that P311 downregulates TGF-β1 and 2 in part by blocking TGF-β autoinduction. [Copyright &y& Elsevier]

Hormones can exert significant protective effects on autoimmune diseases by activating immunoregulatory mechanisms. One of the possible mechanisms of hormonal protection might be through the anti-inflammatory effects of the TGF-beta molecule. The present study investigated the changes in expression of two TGF-beta isoforms, TGF-β1 and TGF-β3, in C57BL/6 and TCR transgenic (T/R+) B10.PL mice that manifested or were protected against clinical signs of experimental autoimmune encephalomyelitis (EAE) with 17β-estradiol (E2) treatment. We here demonstrate an inverse relationship between expression of TGF-β1 that is enhanced in mice with EAE, and TGF-β3 that is enhanced in E2-protected mice. The differential expression of TGF-β isoforms was observed in spinal cord tissue but not spleen. Additionally TGF-β1 expression was evident both in whole spinal cord tissue and mononuclear cells isolated from inflamed tissue, in contrast to TGF-β3 that was only detected in spinal cord tissue but not in mononuclear cells. Further studies revealed that CD3 and especially MAC-1 positive cells were the main source of TGF-β1 in the mononuclear CNS population. Of crucial importance, the TGF-β3 isoform displayed anti-proliferative properties towards encephalitogenic cells in vitro. We propose that the TGF-β1 and TGF-β3 isoforms play opposing roles in the expression of EAE. [Copyright &y& Elsevier]

Palate fusion is a complex process that involves the coordination of a series of cellular changes including cell death and epithelial to mesenchymal transition (EMT). Since members of the Snail family of zinc-finger regulators are involved in both triggering of the EMT and cell survival, we decided to study their putative role in palatal fusion. Furthermore, Snail genes are induced by transforming growth factor β gene (TGF-β) superfamily members, and TGF-β3 null mutant mice (TGF-β3−/−) show a cleft palate phenotype. Here we show that in the wild-type mouse at the time of fusion, Snail is expressed in a few cells of the midline epithelial seam (MES), compatible with a role in triggering of the EMT in a small subpopulation of the MES. We also find an intriguing relationship between the expression of Snail family members and cell survival associated to the cleft palate condition. Indeed, Snail is expressed in the medial edge epithelial (MEE) cells in TGF-β3−/−mouse embryo palates, where it is activated by the aberrant expression of its inducer, TGF-β1, in the underlying mesenchyme. In contrast to Snail-deficient wild-type pre-adhesion MEE cells, Snail-expressing TGF-β3 mutant MEE cells survive as they do their counterparts in the chick embryo. Interestingly, Slug is the Snail family member expressed in the chick MEE, providing another example of interchange of Snail and Slug expression between avian and mammalian embryos. We propose that in the absence of TGF-β3, TGF-β1 is upregulated in the mesenchyme, and that in both physiological (avian) and pathological (TGF-β3−/−mammalian) cleft palates, it induces the expression of Snail genes promoting the survival of the MEE cells and permitting their subsequent differentiation into keratinized stratified epithelium. [Copyright &y& Elsevier]

Objective To compare the mRNA levels of transforming growth factor beta (TGF-β) isoforms 1, 2, and 3 among surgically injured peritoneum that healed with adhesions, surgically injured peritoneum that healed without adhesions, and uninjured peritoneum. Design Prospective experimental study. Setting University vivarium. Animal(s) Fourteen sexually mature female Sprague-Dawley rats, 226–250 grams. Interventions(s) Standardized cecal abrasion was performed on 120 Sprague-Dawley rats. The areas of abrasion and any resultant adhesions were harvested at necropsy 7 days later. Total RNA was then extracted from the serosal adhesions of four rats, from the serosa of five rats that healed without adhesion formation, and from analogous areas of cecum from five rats that did not undergo abrasion and served as controls. Main outcome measure(s) Quantification of the expression of the TGF-β1, TGF-β2, and TGF-β3 mRNA transcripts in peritoneal adhesions, normally healed peritoneum, and fresh, uninjured peritoneal tissue through the use of multiplex reverse transcriptase–polymerase chain reaction analysis. Results(s) Peritoneal adhesion TGF-β1 and TGF-β3 mRNA expression was 3.7-fold and 8.6-fold higher, respectively, than in abraded tissues that healed normally; and 5.6-fold and 4.6-fold higher, respectively, than in nonabraded tissues. While TGF-β2 mRNA levels were also higher in serosal adhesions compared with normally healed and uninjured peritoneum, this rise was not statically significant. Conclusion(s) Peritoneal adhesions resulting from surgical abrasion of a serosal surface have statistically significant increased levels of TGF-β1 and -β3 mRNA transcripts compared with both uninjured and normally healed peritoneum. [ABSTRACT FROM AUTHOR]

Background. Transforming growth factor-β3 is a cytokine which is involved in cell growth regulation and differentiation, stimulation of extracellular matrix and modulation of immune responses. The goal of this study was to detect the presence of this cytokine in the myometrium of preterm and term, nonlaboring and laboring patients, and to measure serum levels of interleukin-1β (IL-1β), IL-6 and IL-8 before cesarean section. Methods. In this prospective study, we obtained samples of myometrium from the lower uterine segment during elective and emergency cesarean sections (term non-laboring, n=8; term laboring, n=7; preterm non-laboring, n=3; and preterm laboring, n=19) and stained for transforming growth factor-β3. Blood was also sampled from the same patients to determine IL-1β, IL-6 and IL-8 levels. Results. Different intensities of staining were detected in preterm laboring, term nonlaboring and term laboring groups, but there was no staining in preterm nonlaboring group. We also found a statistically significant difference in IL-6 levels between laboring and nonlaboring groups (p=0.028). Conclusion. Different intensities of TGF-β3 which appeared in different stages of myometrium made us consider that TGF-β3 might prepare myometrium to labor, and IL-6 was more important than the other interleukins in initiation of labor. [ABSTRACT FROM AUTHOR]

Elevated local expression of transforming growth factor (TGF-β) has been associated with increased incidence of peritoneal adhesion formation. In this study we determine whether differences in basal expression of TGF-β in serosal tissue of peritoneal organs correlate with incidence of adhesion formation. Serosal tissue of parietal peritoneum, uterus, oviduct, ovary, omentum, large and small bowels as well as adhesions, skin, fascia, subcutaneous tissue, peritoneal fluid and serum were collected from 57 subjects with/without adhesions who were undergoing abdominal/pelvic surgery. To determine TGF-β1 and TGF-β3 mRNA and protein expression, total RNA and protein were isolated from these tissues and along with the fluids, subjected to quantitative RT–PCR and enzyme-linked immunosorbent assay (ELISA) respectively. Tissue sections were immunostained for TGF-β1 and TGF-β3 protein. We found that TGF-β1 and TGF-β3 mRNA and protein are expressed in these tissues and present in peritoneal fluids and serum, with considerable variations in level of their expression. Comparatively, there was more variation in TGF-β1 than TGF-β3 expression without age or gender relation. Adhesions express a significantly higher TGF-β1 mRNA and have the highest TGF-β1:TGF-β3 ratio, with lowest concentrations and ratio detected in omentum, small and large bowels; in contrast uterus expresses higher TGF-β3, with lowest concentrations detected in subcutaneous tissue and large bowels (P < 0.05). A similar trend was also observed for total (active + latent) TGF-β1 protein expression, with low active TGF-β1 that was not significantly different among the tissue extracts and fluids. However, the lowest active:total TGF-β1 ratio was found in adhesions and ovary. In subjects with adhesions, the adhesions express significantly more TGF-β1 compared to parietal peritoneum (P < 0.05). Immunoreactive TGF-β1 and TGF-β3 protein were present in various cell types in these tissues with intensity reflecting their mRNA and protein expression. In conclusion, we provided evidence that serosal tissue of various peritoneal organs and adhesions express TGF-β1 and TGF-β3. Since TGF-β is expressed differently in these tissues and tissue injury often alters the expression of TGF-β, we propose that tissues with a higher basal expression of TGF-β may become predisposed to develop more adhesions compared to others. [ABSTRACT FROM PUBLISHER]

Background: The Autologous Chondrocytes Transplantation (ACT) method is being studied for repair of cartilage diseases. As the chondrocytes dedifferentiated during monolayer culture, three-dimensional cultures are suggested to redifferentiate them. The aim of this study was investigation of the effect of TGF-β3 growth factor on chondrocytes in pellet culture system. Materials and Methods: The chondrocytes were isolated from three human articular cartilages by enzymatic digestion. The cells of the second passage were transferred to pellet culture system. We determined the chondrogenic medium with TGF-β3 as the experimental group and without it as the control group. After 2 weeks, the aggrecan production was investigated using histological and immunohistochemical (IHC) methods. Results: The presence of glycosaminoglycans was proved through Toluiden blue staining. Comparison of IHC results using MATLAB software showed that aggrecan in the experimental group was significantly higher than in the control group (P ≤ 0.05). Conclusion: The presence of TGF-β3 in the chondrogenic medium could lead to the production of more aggrecan in chondrocytes cultivated in pellet culture system.

Articular cartilage (AC) damage is quite common, but due to AC's poor self-healing ability, the damage can easily develop into osteoarthritis (OA). To solve this problem, we developed a microsphere/hydrogel system that provides two growth factors that promote cartilage repair: transforming growth factor-β3 (TGF-β3) to enhance cartilage tissue formation and ghrelin synergy TGF-β to significantly enhance the chondrogenic differentiation. The hydrogel and microspheres were characterized in vitro, and the biocompatibility of the system was verified. Double emulsion solvent extraction technology (w/o/w) is used to encapsulate TGF-β3 and ghrelin into microspheres, and these microspheres are encapsulated in a hydrogel to continuously release TGF-β3 and ghrelin. According to the chondrogenic differentiation ability of mesenchymal stem cells (MSCs) in vitro, the concentrations of the two growth factors were optimized to promote cartilage regeneration. [ABSTRACT FROM AUTHOR]

Cleft palate is one of the major congenital craniofacial birth defects. The etiology underlying the pathogenesis of cleft palate has yet to be fully elucidated. Dissociation of the medial edge epithelium (MEE) at the contacting region of palatal shelves and subsequent migration or apoptosis of MEE cells is required for proper MEE removal. Ras-responsive element-binding protein 1 (RREB1), a RAS transcriptional effector, has recently been shown to play a crucial role in developmental epithelial-mesenchymal transition (EMT), in which loss of epithelial characteristics is an initial step, during mid-gastrulation of embryonic development. Interestingly, the involvement of RREB1 in cleft palate has been indicated in humans. Here, we demonstrated that pan-Ras inhibitor prevents the dissociation of MEE during murine palatal fusion. Rreb1 is expressed in the palatal epithelium during palatal fusion, and knockdown of Rreb1 in palatal organ culture resulted in palatal fusion defects by inhibiting the dissociation of MEE cells. Our present findings provide evidence that RREB1-mediated Ras signaling is required during palatal fusion. Aberrant RREB1-mediated Ras signaling might be involved in the pathogenesis of cleft palate., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2022. Published by The Company of Biologists Ltd.)

Background: Organogenesis from tonsil-derived mesenchymal cells (TMSCs) has been reported, wherein tenogenic markers are expressed depending on the chemical stimulation during tenogenesis. However, there are insufficient studies on the mechanical strain stimulation for tenogenic cell differentiation of TMSCs, although these cells possess advantages as a cell source for generating tendinous tissue. The purpose of this study was to investigate the effects of mechanical strain and transforming growth factor-beta 3 (TGF-β3) on the tenogenic differentiation of TMSCs and evaluate the expression of tendon-related genes and extracellular matrix (ECM) components, such as collagen., Results: mRNA expression of tenogenic genes was significantly higher when the mechanical strain was applied than under static conditions. Moreover, mRNA expression of tenogenic genes was significantly higher with TGF-β3 treatment than without. mRNA expression of osteogenic and chondrogenic genes was not significantly different among different mechanical strain intensities. In cells without TGF-β3 treatment, double-stranded DNA concentration decreased, while the amount of normalized collagen increased as the intensity of mechanical strain increased., Conclusions: Mechanical strain and TGF-β3 have significant effects on TMSC differentiation into tenocytes. Mechanical strain stimulates the differentiation of TMSCs, particularly into tenocytes, and cell differentiation, rather than proliferation. However, a combination of these two did not have a synergistic effect on differentiation. In other words, mechanical loading did not stimulate the differentiation of TMSCs with TGF-β3 supplementation. The effect of mechanical loading with TGF-β3 treatment on TMSC differentiation can be manipulated according to the differentiation stage of TMSCs. Moreover, TMSCs have the potential to be used for cell banking, and compared to other mesenchymal stem cells, they can be procured from patients via less invasive procedures., (© 2021. The Author(s).)

The objective of this study was to determine the expression and functional role of a long noncoding RNA (lncRNA) MIAT (myocardial infarction-associated transcript) in leiomyoma pathogenesis. Leiomyoma compared with myometrium (n = 66) expressed significantly more MIAT that was independent of race/ethnicity and menstrual cycle phase but dependent on MED12 (mediator complex subunit 12) mutation status. Leiomyomas bearing the MED12 mutation expressed higher levels of MIAT and lower levels of microRNA 29 family (miR-29a, -b, and -c) compared with MED12 wild-type leiomyomas. Using luciferase reporter activity and RNA immunoprecipitation analysis, MIAT was shown to sponge the miR-29 family. In a 3-dimensional spheroid culture system, transient transfection of MIAT siRNA in leiomyoma smooth muscle cell (LSMC) spheroids resulted in upregulation of miR-29 family and downregulation of miR-29 targets, collagen type I (COL1A1), collagen type III (COL3A1), and TGF-β3 (transforming growth factor β-3). Treatment of LSMC spheroids with TGF-β3 induced COL1A1, COL3A1, and MIAT levels, but repressed miR-29 family expression. Knockdown of MIAT in LSMC spheroids blocked the effects of TGF-β3 on the induction of COL1A1 and COL3A1 expression. Collectively, these results underscore the physiological significance of MIAT in extracellular matrix accumulation in leiomyoma., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Endocrine Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Osteoarthritic degeneration of cartilage is a major social health problem. Tissue engineering of cartilage using combinations of scaffold and mesenchymal stem cells (MSCs) is emerging as an alternative to existing treatment options such as microfracture, mosaicplasty, allograft, autologous chondrocyte implantation, or total joint replacement. Induction of chondrogenesis in high-density pellets of MSCs is generally attained by soluble exogenous TGF-β3 in culture media, which requires lengthy in vitro culture period during which pellets gain mechanical robustness. On the other hand, a growth factor delivering and a mechanically robust scaffold material that can accommodate chondroid pellets would enable rapid deployment of pellets after seeding. Delivery of the growth factor from the scaffold locally would drive the induction of chondrogenic differentiation in the postimplantation period. Therefore, we sought to develop a biomaterial formulation that will induce chondrogenesis in situ, and compared its performance to soluble delivery in vitro . In this vein, a heparin-conjugated mechanically robust collagen fabric was developed for sustained delivery of TGF-β3. The amount of conjugated heparin was varied to enhance the amount of TGF-β3 uptake and release from the scaffold. The results showed that the scaffold delivered TGF-β3 for up to 8 days of culture, which resulted in 15-fold increase in GAG production, and six-fold increase in collagen synthesis with respect to the No TGF-β3 group. The resulting matrix was cartilage like, in that type II collagen and aggrecan were positive in the spheroids. Enhanced chondrogenesis under in situ TGF-β3 administration resulted in a Young's modulus of ∼600 kPa. In most metrics, there were no significant differences between the soluble delivery group and in situ heparin-mediated delivery group. In conclusion, heparin-conjugated collagen scaffold developed in this study guides chondrogenic differentiation of hMSCs in a mechanically competent tissue construct, which showed potential to be used for cartilage tissue regeneration. Impact statement The most significant finding of this study was that sustained release of TGF-β3 from heparinized collagen scaffold had chondroinductive effect on pelleted human mesenchymal stem cells (hMSCs). The effect was comparable to that observed in hMSC pellets that were cultured in chondrogenic media supplemented with TGF-β3. The stiffness of scaffolds at the baseline was about 50% that of native cartilage and over 28 days the combined stiffness of pellet/scaffold complex converged to the stiffness of native cartilage. These data indicate that the scaffold system can generate a load-bearing cartilage-like tissue by using hMSCs pellets in a mechanically competent framework.

Background: The injured flexor tendon has poor healing ability, which is easy to cause tendon adhesion. It can affect the recovery of tendon function, which is still a long-term and difficult task for surgeons. Transforming growth factor β (TGF-β) has been widely considered to play an important role in flexor tendon repair in recent years., Aim: This work was to investigate the anti-adhesion and anti-inflammatory effects of TGF-β3 on flexor digitorum longus (FDL) tendon repair rats., Method: Anastomosis models of tendon laceration in the flexion toes of rats were delivered with no treatment, vehicle, or TGF-β3 -overexpressed adenovirus vector (ad-TGF-β3) locally to the injured tendon area from day 3 to 8. Subsequently, the expression of TGF-β3, TGF-β1/2, Smad3, Smad7, JNK, phosphorylation (p)-JNK, c-Jun, and phosphorylation (p)-c-Jun were detected by western blot, the expression of Mmp9 and Mmp2 by RT-qPCR, the Range of motion (ROM) and gliding resistance by adhesion formation testing, the mechanical strength of tendon healing by biomechanical testing, the pathologic changes of flexor tendon tissues by HE staining, the expression of collagen type III by immunohistochemical staining, and the levels of IL-6, TNF-α, COX2 and IL-1β in serum by ELISA, respectively., Results: Rat models treated with no treatment showed a lower elevation of TGF-β3 and Smad7 expression, and a higher elevation of TGF-β1/2 and Smad3 expression, during day 14 to day 28. Besides, under the treatment of ad-TGF-β3, a significantly increase was reflected in the expression of TGF-β3 and Smad7, ROM, as well as mechanical strength of flexor tendon, whereas significantly reduction was shown in gliding resistance, the content of inflammatory cytokines, the ratio of p-JNK/JNK, p-c-Jun/c-Jun, as well as the expression of TGF-β1/2, Smad3, Mmp9, and Mmp2 genes, as compared to those from vehicle treatment. Meanwhile, TGF-β3 demonstrated a better pathologic recovery process with no obvious necrosis or fracture of collagen fibers. Besides, TGF-β3 revealed a significant reduction of collagen type-III expression in the flexor tendon healing tissues., Conclusion: These findings suggested that TGF-β3 effectively protected against flexor tendon injury via regulating adhesion formation., (© 2021. The Author(s).)

2,3,7,8-tetrachlorodibenzo- p -dioxin (TCDD) is a persistent organic pollutant that is strongly associated with a number of human diseases and birth defects, including cleft palate. Transforming growth factor (TGF) plays a significant role during mammalian palatogenesis. However, the epigenetic mechanism of transforming growth factors in the process of TCDD-induced cleft palate is unclear. The purpose of this research was to investigate the relationship and potential mechanism between TGF-β2/3 promoter DNA methylation and Smad signaling during TCDD-induced cleft palate. Pregnant C57BL/6N mice were exposed to 64 µg/kg TCDD on gestational day 10 (GD10) to establish the cleft palate model and palatal tissues of embryos were collected on GD13, GD14, and GD15 for subsequent experiments. TGF-β2/3 mRNA expression, TGF-β2/3 promoter methylation, and Smad signaling molecules expression were assessed in the palate of the two groups. The results showed that the incidence of cleft palate was 94.7% in the TCDD-treated group whereas no cleft palate was found in the control group. TCDD-treated group altered specific CpG sites of TGF-β2/3 promoter methylation. Compared to the control group, the proliferation of mouse embryonic palate mesenchymal stromal cells (MEPM), the expressions of TGF-β2/3, p-Smad2, and Smad4 were all reduced, while the expression of Smad7 was significantly increased in the atAR group. Smad signaling was downregulated by TCDD. Therefore, we suggest that TGF-β2/3 promoter methylation and Smad signaling may be involved in TCDD-induced cleft palate formation in fetal mice.

Varicocele is an age-related disease with no current medical treatments positively impacting infertility. Toll-like receptor 4 (TLR4) expression is present in normal testis with an involvement in the immunological reactions. The role of peroxisome proliferator-activated receptor-α (PPAR-α), a nuclear receptor, in fertility is still unclear. N-Palmitoylethanolamide (PEA), an emerging nutraceutical compound present in plants and animal foods, is an endogenous PPAR-α agonist with well-demonstrated anti-inflammatory and analgesics characteristics. In this model of mice varicocele, PPAR-α and TLR4 receptors' roles were investigated through the administration of ultra-micronized PEA (PEA-um). Male wild-type (WT), PPAR-α knockout (KO), and TLR4 KO mice were used. A group underwent sham operation and administration of vehicle or PEA-um (10 mg/kg i.p.) for 21 days. Another group (WT, PPAR-α KO, and TLR4 KO) underwent surgical varicocele and was treated with vehicle or PEA-um (10 mg/kg i.p.) for 21 days. At the end of treatments, all animals were euthanized. Both operated and contralateral testes were processed for histological and morphometric assessment, for PPAR-α, TLR4, occludin, and claudin-11 immunohistochemistry and for PPAR-α, TLR4, transforming growth factor-beta3 (TGF-β3), phospho-extracellular signal-Regulated-Kinase (p-ERK) 1/2, and nucleotide-binding oligomerization domain-like receptor (NLR) family pyrin domain-containing 3 (NLRP3) Western blot analysis. Collectively, our data showed that administration of PEA-um revealed a key role of PPAR-α and TLR4 in varicocele pathophysiology, unmasking new nutraceutical therapeutic targets for future varicocele research and supporting surgical management of male infertility. [ABSTRACT FROM AUTHOR]

TGF-β3 has been reported to have a strong therapeutic efficacy in wound healing when externally administered, but TGF-β3's active form is rapidly metabolized and removed from the body. Therefore, a drug delivery system that can provide a new non-toxic and an effective treatment that could be locally applied and also be able to protect the stability of the protein and provide controlled release is required. The aim of the study is to prepare and characterize nanoparticles and nanostructured films with TGF-β3 and to evaluate in vitro cytotoxicity of the loaded nanoparticles. PCL-based films containing TGF-β3 or TGF-β3-loaded PLGA nanoparticles were prepared with non-toxic modified solvent displacement method. The particle size and protein loading efficiency of TGF-β3-loaded PLGA nanoparticles were 204.9 ± 10.3 nm and 42.42 ± 2.03%, respectively. In vitro release studies of TGF-β3-loaded PLGA nanoparticle formulations revealed that the protein was completely released from the nanoparticles at the end of 24 h. In vitro release profile of film formulation containing TGF-β3-loaded nanoparticles was similar. TGF-β3 released from nanoparticles do not have a significant effect on proliferation of HepG2 cells demonstrating their biocompatibility. Additionally, prepared films were tested with in vivo wound healing mouse model and showed to heal significantly faster and with improved scarring. PCL films loaded with TGF-β3 or TGF-β3 nanoparticles prepared in this study may be an effective treatment approach for wound healing therapy after injury., (© 2021. American Association of Pharmaceutical Scientists.)

Cyclophosphamide (CP) is commonly used as an anticancer agent but has been associated with high toxicity in several organs, including the testes. In this study, we aimed to evaluate the effects of CP-induced testicular toxicity, using glial cell line-derived neurotrophic factor (GDNF), occludin and transforming growth factor beta 3 (TGF-β3) primary antibodies, and miR-34b and miR-34c expressions. Eighteen young Balb/c male mice were divided into three groups. The control group received no treatment. The mice of CP group were injected 100 mg kg -1  day -1 CP for 5 days, and the same amount of saline was injected in the sham group. The animals were sacrificed 24 hr after the last injection. Immunohistochemical analysis of testicular tissues showed a decrease in both spermatogenic germ cell count and also GDNF, occludin expressions, but an increase in TGF-β3 expression in the CP group compared to the others group. The expressions of miR-34b and miR-34c were examined by qPCR technique, a significant decrease was observed in tissue samples in the CP-treated group. The expression of GDNF, occludin and TGF-β3 plays an important role in testicular injury caused by CP, and the decrease in the expression of miR-34b/c in tissue samples may be an important marker for the detection of testicular damage., (© 2021 Wiley-VCH GmbH.)

Low back pain caused by intervertebral disc degeneration has become a global problem that seriously affects public health. The application of nucleus pulposus tissue engineering to disc degeneration has attracted increasing attention. A scaffold is important for nucleus pulposus tissue engineering, which provides a three-dimensional growth space with an appropriate biomechanical and biochemical microenvironment for seed cell differentiation and proliferation. In this study, a decellularized nucleus pulposus matrix/chitosan (DNPM/chitosan) hydrogel scaffold was prepared with crosslinker genipin. Nucleus pulposus stem cells (NPSCs) were cultured in hybrid hydrogels with or without transforming growth factor-β3 (TGF-β3) and then cell morphology, proliferation, and nucleus pulposus-related gene expression were analyzed. TGF-β3 was successfully incorporated into the DNPM/chitosan hydrogel and NPSCs grew well on both kinds of hydrogel. Moreover, gene expression of collagen-I, collagen-II, and aggrecan was enhanced in the DNPM/chitosan hydrogel with TGF-β3. These results indicate that the DNPM/chitosan hybrid hydrogel is a promising candidate scaffold for nucleus pulposus tissue engineering., Competing Interests: Conflict of interestThe authors declare no conflicts of interest., (© The Author(s), under exclusive licence to Springer Nature B.V. 2021.)