Cleft palate is a common birth defect in humans. Elevation and fusion of paired palatal shelves are coordinated by growth and transcription factors, and mutations in these can cause malformations. Among the effector genes for growth factor signaling are extracellular matrix (ECM) glycoproteins. These provide substrates for cell adhesion (e.g., fibronectin, tenascins), but also regulate growth factor availability (e.g., fibrillins). Cleft palate in Bmp7 null mouse embryos is caused by a delay in palatal shelf elevation. In contrast, palatal shelves of Tgf-β3 knockout mice elevate normally, but a cleft develops due to their failure to fuse. However, nothing is known about a possible functional interaction between specific ECM proteins and Tgf-β/Bmp family members in palatogenesis. To start addressing this question, we studied the mRNA and protein distribution of relevant ECM components during secondary palate development, and compared it to growth factor expression in wildtypewild type and mutant mice. We found that fibrillin-2 (but not fibrillin-1) mRNA appeared in the mesenchyme of elevated palatal shelves adjacent to the midline epithelial cells, which were positive for Tgf-β3 mRNA. Moreover, midline epithelial cells started expressing fibronectin upon contact of the two palatal shelves. These findings support the hypothesis that fibrillin-2 and fibronectin are involved in regulating the activity of Tgf-β3 at the fusing midline. In addition, we observed that tenascin-W (but not tenascin-C) was misexpressed in palatal shelves of Bmp7-deficient mouse embryos. In contrast to tenascin-C, tenascin-W secretion was strongly induced by Bmp7 in embryonic cranial fibroblasts in vitro. These results are consistent with a putative function for tenascin-W as a target of Bmp7 signaling during palate elevation. Our results indicate that distinct ECM proteins are important for morphogenesis of the secondary palate, both as downstream effectors and as regulators of Tgf-β/Bmp activity.

Heparan sulfate (HS) interacts with growth factors and has been implicated in regulating chondrogenesis. However, the effect of HS on TGF-β-mediated mesenchymal stem cell (MSC) chondrogenesis and molecular mechanisms remains unknown. In this study, we explored the effects of exogenous HS alone and in combination with TGF-β3 on chondrogenic differentiation of human MSCs and possible signal mechanisms. The results indicated that HS alone had no obvious effects on chondrogenic differentiation of human MSCs and TGF-β/Smad2/3 signal pathways. However, the combined TGF-β3/HS treatment resulted in a significant increase in GAG synthesis, cartilage matrix protein secretion, and cartilage-specific gene expression compared to cells treated with TGF-β3 alone. Furthermore, HS inhibited type III TGF-β receptors (TβRIII) expression and increased TGF-β3-mediated ratio of the type II (TβRII) to the type I (TβRI) TGF-β receptors and phosphorylation levels of Smad2/3. The inhibitor of the TGF-β/Smad signal, SB431542, not only completely inhibited HS-stimulated TGF-β3-mediated Smad2/3 phosphorylation but also completely inhibited the effects of HS on TGF-β3-induced chondrogenic differentiation. These results demonstrate exogenous HS enhances TGF-β3-induced chondrogenic differentiation of human MSCs by activating TGF-β/Smad2/3 signaling.

Transforming growth factor-β (TGF-β) is the prototype of a family of secreted polypeptide growth factors. These cytokines play very important roles during development, as well as in normal physiological and disease processes, by regulating a wide array of cellular processes, such as cell growth, differentiation, migration, apoptosis, and extracellular matrix production. TGF-β utilizes a multitude of intracellular signalling pathways in addition to Smads with actions that are dependent on circumstances, including dose, target cell type, and context. The aims of this research were (i) to verify the effects of dose-dependent TGF-β3 treatment on YY1 and p53 expression, in BPH-1 cell line, human benign prostate hyperplasia, and two prostate cancer cell lines, LNCaP, which is androgen-sensitive, and DU-145, which is androgen-non responsive, (ii) establish a correlation between p53 and YY1 and (iii) determine the expression of a number of important intracellular signalling pathways in TGF-β3-treated prostate cell lines. The expression of YY1, p53, PI3K, AKT, pAKT, PTEN, Bcl-2, Bax, and iNOS was evaluated through Western blot analysis on BPH-1, LNCaP, and DU-145 cultures treated with 10 and 50 ng/ml of TGF-β3 for 24 h. The production of nitric oxide (NO) was determined by Griess reagent and cell viability through MTT assay. The results of this research demonstrated profound differences in the responses of the BPH-1, LNCaP, and DU-145 cell lines to TGF-β3 stimulation. We believe that the findings could be important because of the clinical relevance that they may assume and the therapeutic implications for TGF-β treatment of prostate cancer.

Prostate cancer is the most common type of cancer in men. It is assumed that the tumor microenvironment of the prostate contributes to invasion and metastasis. Stroma-epithelial crosstalk has shown to change with progression of prostate cancer, and thereby the stromal compartment might be an attractive target in diagnostic and therapeutic approaches to prostate cancer. The purpose of this project was to study the reciprocal influence between fibroblasts and cancer cells in prostate cancer. Prostate fibroblast primary cultures from areas with cancer and hyperplasia were cocultivated with cells of the PC-3 lineage. Gene expression profiles of both cell types were studied to reveal possible associations to cancer invasion and metastasis. There were 383 differentially expressed genes between fibroblasts from cancerous areas and fibroblasts from areas with hyperplasia before cocultivation with PC-3 cells. Several of the differentially expressed gene classes are associated with cancer development and metastasis. After cocultivation, there were 26 differentially expressed genes between cancerous and hyperplastic fibroblasts. There were only three differentially expressed genes between PC-3 cells that had been cocultivated with cancerous fibroblasts and PC-3 cells that had been cocultivated with hyperplastic fibroblasts. The fibroblasts from cancer areas showed a different expression pattern from the characteristics reported as reactive stroma in previous studies. We found tenascin C to be downregulated, which is contrary to previous findings. TGF-β3 and TGF-βR3 were also downregulated, which has been associated with disturbance of TGF-β signaling during prostate cancer progression. Cocultivation with PC-3 cells seems to make the cancerous and hyperplastic fibroblasts more alike each other, as the number of differentially expressed genes decreases. It is desirable to find out if the reduction in differential gene expression is attributable to that hyperplastic fibroblasts become more alike the cancerous fibroblasts or vice versa. Also, we think that the lower expression levels of c-Jun and c-Fos in cancerous fibroblasts without coculture may cause loss of normal fibroblast differentiation, proliferation and inflammatory response, and hence, favor the proliferation and invasion of cancer cells.

BACKGROUND

The osteogenic potential of human adipose-derived stromal cells (hASCs), the ease of cell procurement, and the shortcomings of conventional skeletal reconstruction call for further analysis of the molecular mechanisms governing hASC osteogenic differentiation. We have examined the expression profile of the human transcriptome during osteogenic differentiation of ASCs using microarray. Subsequently, we analyzed those genes related to osteogenesis that have not been previously studied about hASCs. We have preliminarily assessed the role of IGFBP3, TGF-B3, TNC, CTGF, DKK-1, and PDGFRB in hASC osteogenic differentiation.

METHODS

We compared the expression profile of undifferentiated hASCs to that of hASCs treated with osteogenic differentiation medium for 1, 3, or 7 days using the Human Exonic Evidence-Based Oligonucleotide chip. Genes significantly overexpress or underexpressed were validated with quantitative reverse transcription-polymerase chain reaction. The osteogenic capability of ASCs was verified by Alizarin Red staining.

RESULTS

IGFBP3, TGF-B3, TNC, CTGF, and PDGFRB were all upregulated in early osteogenesis, and TGF-B3, TNC, and PDGFRB were upregulated in late osteogenesis by microarray and quantitative reverse transcription analysis. In contrast, DKK-1 was downregulated in early and late osteogenesis. Alizarin Red staining showed a significant increase in mineralization in hASCs, even after 1 day in osteogenic differentiation medium.

CONCLUSIONS

Factors that commit hASCs to an osteogenic pathway remain largely unknown. We have described 6 genes that play key roles in hASC osteogenic differentiation. We plan to further exploit these data via in vitro treatment of hASCs with these soluble cytokines and in vivo translation using a nude mouse calvarial defect model.

The elevation of transforming growth factor-β2 (TGF-β2) levels in eye tissue is considered as one of the major factors contributing to posterior capsule opacification (PCO) in patients undergoing cataract surgery, since TGF-β2 is known to stimulate the cell migration of residual human lens epithelial cells (HLECs). The present study aimed to test the potential effect of dexamethasone (DEX) on TGF-β2-induced cell migration and the possible cellular mechanisms involved in this process. Cultured HLE-B3 cells were treated with TGF-β2 (0.1 ng/ml) in the presence or absence of DEX (100 nM). HLE-B3 cell migration was determined by the Phagokinetic Track Motility Assay. Activation of mitogen-activated protein kinase (MAPK) signaling pathways was determined by Western blotting using specific phosphorylation antibodies, matrix metalloproteinase (MMP)-2 and MMP-9 mRNA expression, and activities were analyzed by RT-PCR and gelatin zymography assay, respectively. In cultured HLE-B3 cells, DEX largely inhibited TGF-β2-induced cell migration and MMP activity, probably by inhibiting the ERK/MAPK pathway. We suggest that the use of DEX may be of help in the prevention of PCO formation and development.

Mimicking the native tissue microenvironment is critical for effective tissue regeneration. Mechanical cues and sustained biological cues are important factors, particularly in load-bearing tissues such as articular cartilage or bone. Carriers including hydrogels and nanoparticles have been investigated to achieve sustained release of protein drugs. However, it is difficult to apply such carriers alone as scaffolds for cartilage regeneration because of their weak mechanical properties, and they must be combined with other biomaterials that have adequate mechanical strength. In this study, we developed the multifunctional scaffold which has similar mechanical properties to those of native cartilage and encapsulates TGF-β3 for chondrogenesis. In our previous work, we confirmed that poly(lactide-co-caprolacton) (PLCL) did not foam when exposed to supercritical CO2 below 45°C. Here, we used a supercritical carbon dioxide (scCO2)-1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) co-solvent system to facilitate processing under mild conditions because high temperature causes protein denaturation and decreases bioactivity of the protein. This processing made it possible to fabricate a TGF-β3 encapsulated elastic porous PLCL scaffold at 37°C. We investigated the tissue regeneration efficiency of the TGF-β3 encapsulated PLCL scaffold using human adipose-derived stem cells (ADSCs) in vitro and in vivo (Groups; i. PLCL scaffold+Fibrin gel+TGF-β3, ii. TGF-β3 encapsulated PLCL scaffold+Fibrin gel, iii. TGF-β3 encapsulated PLCL scaffold). We evaluated the chondrogenic abilities of the scaffolds at 4, 8, and 12weeks after subcutaneous implantation of the constructs in immune-deficient mice. Based on TGF-β3 release studies, we confirmed that TGF-β3 molecules were released by 8weeks and remained in the PLCL matrix. Explants of TGF-β3 encapsulated scaffolds by a co-solvent system exhibited distinct improvement in the compressive E-modulus and deposition of extracellular matrix. Furthermore, long-term delivery of TGF-β3 formed a hyaline cartilage-specific lacunae structure and prevented the hypertrophy of differentiated chondrocytes. TGF-β3 encapsulated PLCL scaffolds would be useful as functional scaffolds for cartilage tissue engineering.

OBJECTIVE

The boar, as human, sequentially ejaculates sperm-rich and sperm-poor fractions. Seminal plasma (SP) spermadhesins (PSP-I/PSP-II) induce a primary endometrial inflammatory response in female sows, similar to that elicited by semen deposition in other species, including human. However, the SP is also known to mitigate such response, making it transient to allow for embryo entry to a cleansed endometrium. Although cytokine involvement has been claimed, the exploration of cytokines in different SP fractions is scarce. This study determines Th1, Th2, Th17 and Th3 cytokine profiles in specific ejaculate SP fractions from boars of proven fertility.

METHODS

SP samples from the sperm-rich fraction (SRF) and the sperm-poor post-SRF fraction (post-SRF) of manually collected ejaculates from eight boars (four ejaculates per boar) were analysed by commercial multiplex bead assay kits (Milliplex MAP, Millipore, USA) for interferon-γ, interferon gamma-induced protein 10, macrophage-derived chemokine, growth-regulated oncogene, granulocyte-macrophage colony-stimulating factor, monocyte chemo-attractant protein-1, interleukins (IL)-6, IL-8, IL-10, IL-15, IL-17 and transforming growth factor (TGF)-β1-β3.

RESULTS

Cytokine concentrations differed between the ejaculate fractions among boars, being highest in the post-SRF.

CONCLUSIONS

Boar SP is rich in Th1, Th2, Th17 and Th3 cytokines, with lowest concentrations in the sperm-peak-containing fraction, indicating its main immune influence might reside in the larger, protein-rich sperm-poor post-SRF.

Human intrahepatic cholangiocarcinomas are one of the most difficult cancers to treat. In our study, Lovastatin, a 3-hydroxy-3-methylglutaryl-coenzyme-CoA (HMG-CoA) reductase inhibitor, demonstrated anticancer properties by inhibiting cancer cell proliferation, cell migration and cell adhesion. Lovastatin inhibited the expressions of transforming growth factor (TGF)-β1, cyclooxygenase (COX)-2, and intercellular adhesion molecule (ICAM)-1. Furthermore, lovastatin inhibited the expressions of integrin β1 and integrin β3 but not integrin αv or integrin β5. While Lovastatin's inhibitory effects on TGFβ1, COX2, and ICAM-1 expression were independently controlled by the tumor suppressor LKB1, integrin β3 expression was not affected. Lovastatin's inhibitory effect on cell adhesion was associated with the decreased expression of integrin β3 and cell surface heterodimer integrin αvβ3. Quantitative real time PCR, fluorescent microscopy, and cell migration assays all confirmed that Lovastatin inhibits integrin αvβ3 downstream signaling including FAK activation, and β-catenin, vimentin, ZO-1, and β-actin. Overall, Lovastatin reduced tumor cell proliferation and migration by modifying the expression of genes involved in cell adhesion and other critical cellular processes. Our study highlights novel anti-cancer properties of Lovastatin and supports further exploration of statins in the context of cholangiocarcinoma therapy.

The p53 tumor suppressor protein is primarily known for its important role in tumor suppression. In addition, p53 affects tumor cell migration, invasion, and epithelial-mesenchymal transition (EMT); processes also regulated by the transforming growth factor-β (TGF-β) signaling pathway. Here, we investigated the role of p53 in breast tumor cell invasion, migration, and EMT and examined the interplay of p53 with TGF-β3 in these processes. MCF-10A1 and MCF-10CA1a breast cancer cells were treated with Nutlin-3 and TGF-β3, and the effects on tumor cell migration and invasion were studied in transwell and 3D spheroid invasion assays. The effects of Nutlin-3 and TGF-β3 on EMT were examined in NMuMG cells. To identify genes involved in TGF-β-induced invasion that are modulated by p53, a Human Tumor Metastasis-specific RT-PCR array was performed. Verification of EPHB2 regulation by TGF-β3 and p53 was performed on breast cancer tumor cell lines. We demonstrate that p53 inhibits basal and TGF-β3-induced invasion, migration, and EMT in normal breast epithelial and breast cancer cells. Pharmacological activation of p53 inhibited induction of several TGF-β3 targets involved in TGF-β3-induced tumor cell invasion, i.e., matrix metallo proteinase (MMP)2, MMP9, and integrin β 3 . The ephrin-type B receptor 2 (EPHB2) gene was identified as a new TGF-β target important for TGF-β3-mediated invasion and migration, whose transcriptional activation by TGF-β3 is also inhibited by p53. The results show an intricate interplay between p53 and TGF-β3 whereby p53 inhibits the TGF-β3-induced expression of genes, e.g., EPHB2, to impede tumor cell invasion and migration.

Lysophosphatidic acid (LPA) is a bioactive phospholipid that affects various biological functions, such as cell proliferation, migration, survival, wound healing, and tumor invasion through LPA receptors. Previously, we reported that LPA induces A431 colony dispersal, accompanied by disruption of cell-cell contacts and cell migration. However, it remains unclear how LPA affects cell migration and gene expression during A431 colony dispersal. In this paper, we performed cDNA microarray analysis to investigate this question by comparing gene expression between untreated and LPA-treated A431 cells. Interestingly, these results revealed that LPA treatment upregulates several TGF-β1 target genes, including laminin-332 (Ln-332) components (α3, β3, and γ2 chains). Western blot analysis also showed that LPA increased phosphorylation of Smad2, an event that is carried out by TGF-β1 interactions. Among the genes upregulated, we further addressed the role of Ln-332. Real-time PCR analysis confirmed the transcriptional upregulation of all α3, β3, and γ2 chains of Ln-332 by LPA, corresponding to the protein level increases revealed by western blot. Further, the addition of anti-Ln-332 antibody prevented LPA-treated A431 colonies from dispersing. Taken together, our results suggest that LPA-induced Ln-332 plays a significant role in migration of individual cells from A431 colonies.

Both autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) and the rare thymoma patients with chronic mucocutaneous candidiasis (CMC) have neutralizing autoantibodies to Th17 cytokines and significant defects in production of IL-22 and IL-17F by their T cells. The cause of these defects is unknown. We hypothesized that they might result from autoimmunity against upstream cytokines normally responsible for generating and maintaining Th17 cells.

Luciferase immunoprecipitation (LIPS) was used to screen for autoantibodies to IL-6, IL-1β, TGF-β3, IL-21, and IL-23 in patients with APECED or thymoma. We used Western blotting to assess the conformation-dependence of the IL-6 autoantibodies and flow cytometric analysis of intracellular phospho-STAT3 induction to assess IL-6-neutralizing capacity in IgGs isolated from patient and control sera. We also used Luminex xMAP to measure serum cytokine levels.

We found autoantibodies binding to conformational epitopes of IL-6 in 19.5% of 41 patients with APECED and 12.5% of 104 with thymoma-especially in those with long disease durations. The autoantibodies were predominantly of IgG1 subclass and failed to neutralize IL-6 activity. Notably, serum levels of the IL-6 and IL-17A cytokines were higher in anti-IL-6 seropositive than-negative APECED patients or healthy controls. We also detected autoantibody binding to IL-23 in 27.9% of thymoma patients, resulting from cross-recognition through the p40 subunit it shares with IL-12.

IL-6 and IL-17A elevation in these seropositive patients suggests that antibody-binding may protect IL-6 from degradation and prolong its half-life in vivo.

The formation of new blood vessels from existing vasculature, angiogenesis, is facilitated through a host of different signaling processes. Members of the TGF-β superfamily, TGF-β1, TGF-β3, and BMP9, are key propagators of both inhibition and initiation of angiogenesis. HHT, characterized by AVM and capillary bed defects, is caused by germline mutations in the ENG and ACVRL1/ALK1 genes, respectively. Clinical symptoms include epistaxis and GI hemorrhage. The membranous receptors endoglin and ALK1 activate proliferation and migration of endothelial cells during the angiogenic process via the downstream intracellular SMAD signaling pathway. Endothelial cell senescence or activation is dependent on the type of cytokine, ligand concentration, cell-cell interaction, and a multitude of other signaling molecules. Endoglin and ALK1 receptor levels in tumor vasculature correlate inversely with prognosis in humans, whereas in mice, endoglin deficiency decelerates tumor progression. Therefore, endoglin and ALK1 have been identified as potential therapeutic targets for antibody treatment in various cancers. Early phase clinical trials in humans are currently underway to evaluate the efficacy and safety of biological therapy targeting endoglin/ALK1-mediated cells signaling.

Regeneration of Intervertebral disc (IVD) is a scientific challenge because of the complex structure and composition of tissue, as well as the difficulty in achieving bionic function. Here, an anatomically correct IVD scaffold composed of biomaterials, cells, and growth factors were fabricated via three-dimensional (3D) bioprinting technology. Connective tissue growth factor (CTGF) and transforming growth factor-β3 (TGF-β3) were loaded onto polydopamine nanoparticles, which were mixed with bone marrow mesenchymal stem cells (BMSCs) for regenerating and simulating the structure and function of the nucleus pulposus and annular fibrosus. In vitro experiments confirmed that CTGF and TGF-β3 could be released from the IVD scaffold in a spatially controlled manner, and induced the corresponding BMSCs to differentiate into nucleus pulposus like cells and annulus fibrosus like cells. Next, the fabricated IVD scaffold was implanted into the dorsum subcutaneous of nude mice. The reconstructed IVD exhibited a zone-specific matrix that displayed the corresponding histological and immunological phenotypes: primarily type II collagen and glycosaminoglycan in the core zone, and type I collagen in the surrounding zone. The testing results demonstrated that it exhibited good biomechanical function of the reconstructed IVD. The results presented herein reveal the clinical application potential of the dual growth factors-releasing IVD scaffold fabricated via 3D bioprinting. However, the evaluation in large mammal animal models needs to be further studied.

Keywords: 3D bioprinting; Growth factor (GF); Intervertebral disc (IVD); Mesenchymal stem cells (MSCs); Regenerative medicine.

OBJECTIVE

The aim of this study was to investigate the chondrogenic potential of stem cells from human exfoliated teeth (SHED).

METHODS

SHED cultures were isolated from human exfoliated deciduous teeth. Colony-forming capacity, odonto/osteogenic and adipogenic potential were measured. SHED were cultured for 2 weeks in chondrogenic differentiation medium containing dexamethasone, insulin, ascorbate phosphate, TGF-β3 and bFGF. Toluidine blue staining and safranin O staining were used for chondrogenesis analysis. The related markers, type II collagen and aggrecan, were also investigated using immunohistochemistry. SHED were seeded onto the β-TCP scaffolds and transplanted into the subcutaneous space on the back of nude mice. The transplants were recovered at 2, 4 and 8 weeks post-transplantation for analysis.

RESULTS

SHED showed colony-forming capacity, odonto/osteogenic and adipogenic differentiation capacity. Chondrogenic differentiation was confirmed by toluidine blue staining, safranin O staining, type II collagen and aggrecan immunostaining. After in vivo transplantation, SHED recombined with β-TCP scaffolds were able to generate new cartilage-like tissues.

CONCLUSIONS

The findings demonstrate the chondrogenic differentiation capacity of SHED both in vitro and in vivo models, suggesting the potential of SHED in cartilage tissue engineering.

Different forms of biomaterials, including microspheres, sponges, hydrogels and nanofibres have been broadly used in cartilage regeneration; however, effects of internal structures of biomaterials on chondrogenesis of mesenchymal stem cells (MSCs) remain largely unexplored. Here we investigated the effect of physical microenvironments of sponges and hydrogels on chondrogenic differentiation of MSCs. MSCs, cultured in these two scaffold systems, were induced with TGF-β3 in chondrogeneic differentiation medium and the chondrogenic differentiation was evaluated and compared after three weeks. MSCs in the sponges clustered with spindle morphologies, while they distributed homogenously with round morphologies in the hydrogel. The MSCs proliferated faster in the sponge compared to that in the hydrogel. Significantly higher glycosaminoglycan and collagen II were found in the sponges but not in the hydrogels. The different tissue formation ability of MSCs in these two systems could be attributed to the different metabolic requirements and the cellular events prerequisite in the chondrogenic process of MSCs. It is reasonable to conclude that sponges with relatively active microenvironments that facilitate cell-cell contacts and cell-matrix interaction are optimal for early stage of chondrogeneic differentiation.

The objective of the described experiments was to determine the electrical parameters that lead to optimal expression of a number of bone-related genes in cultured human bone cells exposed to a capacitively coupled electric field. Human calvarial osteoblasts were grown in modified plastic Cooper dishes in which the cells could be exposed to various capacitively coupled electric fields. The optimal duration of stimulation and optimal duration of response to the electrical field, and the optimal amplitude, frequency and duty cycle were all determined for each of the genes analyzed. Results indicated that a capacitively coupled electric field of 60 kHz, 20 mV/cm, 50% duty cycle for 2 h duration per day significantly up-regulated mRNA expression of a number of transforming growth factor (TGF)-β family genes (bone morphogenetic proteins (BMP)-2 and -4, TGF-β1, - β2 and -β3) as well as fibroblast growth factor (FGF)-2, osteocalcin (BGP) and alkaline phosphatase (ALP). Protein levels of BMP-2 and -4, and TGF-β1 and - β2 were also elevated. The clinical relevance of these findings in the context of a noninvasive treatment modality for delayed union and nonunion fracture healing is discussed.

Successful regeneration and remodeling of neuromuscular junctions are critical for restoring functional capacities and properties of skeletal muscle after damage, and axon-guidance molecules may be involved in the signaling that regulates such restoration. Recently, we found that early-differentiated satellite cells up-regulate a secreted neural chemorepellent Sema3A upon in vivo muscle-crush injury. The study also revealed that Sema3A expression is up-regulated in primary satellite-cell cultures in response to hepatocyte growth factor (HGF) and basic fibroblast growth factor (FGF2) and is prevented by transforming growth factor (TGF)-β2, 3. In order to verify the physiological significance of this regulation in vitro, the present study was designed to estimate the time-course of extracellular HGF, FGF2 and TGF-β3 concentrations after crush-injury of Gastrocnemius muscle in the rat lower hind-limb, using a combination of a non-homogenization/non-spin extraction of extracellular wound fluids and enhanced chemiluminescence-Western blotting analyses. Results clearly demonstrated that active HGF and FGF2 are prevalent in 2-8 days post-crush, whereas active TGF-β3 increases after 12 days, providing a better understanding of the time-coordinated levels of HGF, FGF2 and TGF-β3 that drive regulation of Sema3A expression during regenerative intramuscular moto-neuritogenesis.

Cadmium (Cd) causes male infertility. There is the need to identify safe treatments counteracting this toxicity. Flavocoxid is a flavonoid that induces a balanced inhibition of cyclooxygenase (COX)-1 and COX-2 peroxidase moieties and of 5-lipoxygenase (LOX) and has efficacy in the male genitourinary system. We investigated flavocoxid effects on Cd-induced testicular toxicity in mice. Swiss mice were divided into 4 groups: 2 control groups received 0.9% NaCl (vehicle; 1 ml/kg/day) or flavocoxid (20 mg/kg/day ip); 2 groups were challenged with cadmium chloride (CdCl2; 2 mg/kg/day ip) and administered with vehicle or flavocoxid. The treatment lasted for 1 or 2 weeks. The testes were processed for biochemical and morphological studies. CdCl2 increased phosphorylated extracellular signal-regulated kinase (p-ERK) 1/2, tumor necrosis factor (TNF)-α, COX-2, 5-LOX, malondialdehyde (MDA), B-cell-lymphoma (Bcl)-2-associated X protein (Bax), follicle-stimulating hormone (FSH), luteinizing hormone (LH), transforming growth factor (TGF) -β3, decreased Bcl-2, testosterone, inhibin-B, occludin, N-Cadherin, induced structural damages in the testis and disrupted the blood-testis barrier. Many TUNEL-positive germ cells and changes in claudin-11, occludin, and N-cadherin localization were present. Flavocoxid administration reduced, in a time-dependent way, p-ERK 1/2, TNF-α, COX-2, 5-LOX, MDA, Bax, FSH, LH, TGF-β3, augmented Bcl-2, testosterone, inhibin B, occludin, N-Cadherin, and improved the structural organization of the testis and the blood-testis barrier. Few TUNEL-positive germ cells were present and a morphological retrieval of the intercellular junctions was observed. In conclusion, flavocoxid has a protective anti-inflammatory, antioxidant, and antiapoptotic function against Cd-induced toxicity in mice testis. We suggest that flavocoxid may play a relevant positive role against environmental levels of Cd, otherwise deleterious to gametogenesis and tubular integrity.

Uterine fibroids (UFs) are benign tumors of the female genital tract made of the smooth muscle of the uterus. UF growth depends mostly on the influence of the steroid hormones and selected growth factors. Transforming growth factor β (TGF-βs) is a polypeptide that consists of three isoforms: TGF-β1, TGF-β2, and TGF-β3. At present, TGF-β is considered to be one of the key factors in the pathophysiology of UFs. It plays a major role in cellular migration within the tumor, stimulates tumor growth, and enhances tumor metabolism. As a consequence of various dependencies, the synthesis and release of TGF-β in a UF tumor is increased, which results in excessive extracellular matrix production and storage. High concentrations or overexpression of TGF-β mediators may be responsible for clinically symptomatic UFs. The aim of this review was to check the available evidence for the influence of the TGF-β family on UF biology. We conducted their search in PubMed of the National Library of Medicine with the use of the following selected keywords: "uterine fibroid", "leiomyoma", and "transforming growth factor β". After reviewing the titles and abstracts, more than 115 full articles were evaluated. We focused on the TGF-β-related molecular aspects and their influence on the most common symptoms that are associated with UFs. Also, we described how the available data might implicate the current medical management of UFs.

Chondrocytes are the primary candidate therapeutic cells to cure cartilaginous lesions. Ideally, for transplantation, autologous chondrocytes are isolated from the patient, amplified in vitro, seeded in a scaffold and implanted back. However, significant concerns arise with chondrocyte dedifferentiation during monolayer amplification, whereby cells lose their chondrocytic phenotype by rapidly downregulating the expression of cartilage markers such as type II collagen (Col II) and aggrecan. The accompanying upregulation in type I collagen (Col I) is also problematic, as it leads to unexpected fibrosis and causes such engineered cartilage to lack the desired mechanical strength to make up joint lesions. Transforming growth factor-β3 (TGF-β3) has been proved effective in maintaining chondrocytic morphology and promoting total collagen production. In this study, we aimed to deliver the TGF-β3 gene into dedifferentiated chondrocytes with recombinant lentiviral vectors; by transgenic expression of TGF-β3, chondrocytic redifferentiation is catalysed. Simultaneously, shRNA targeting Col I was also incorporated into the vector to suppress Col I production. The results indicated that chondrocytes underwent dedifferentiation in monolayer culture in the presence or absence of transgenic TGF-β3. In three-dimensional culture, effective redifferentiation was managed in the dedifferentiated chondrocytes that were transduced with transgenic TGF-β3. The incorporation and expression of Col I-targeting shRNA were also effective in reducing Col I production in a post-transcriptional manner.

Signaling by members of the transforming growth factor-β (TGF-β) superfamily, such as TGF-β3 and BMP7, and oxygen tension play a pivotal role in chondrocyte biology. The objective of this research was to investigate the endogenous BMP7 expression in human osteoarthritis (OA) cartilage and the effect of oxygen tension on the single or combined treatment with TGF-β3 and BMP7 on OA chondrocyte redifferentiation in three dimensional (3D) pellet cultures. The results showed the expression of BMP7 and its intracellular signaling target SMAD1/5/8 was decreased in early OA, while it was increased in later stages of OA. The combined treatment with TGF-β3 and BMP7, both in normoxia and hypoxia, was more effective than TGF-β3 or BMP7 alone in redifferentiating chondrocytes. This was reflected by Alcian blue/Safranin O staining and collagen type II protein expression, as well as by gene expression. Hypoxia elevated TGF-β3 and BMP7-induced matrix formation of OA chondrocytes and alleviated the catabolic gene expression. Interestingly, cells cultured under normoxia displayed mild signs of an inflammatory stress response, which was effectively counteracted by culturing the cells under low oxygen tension. Our data underscores the important modulatory role of oxygen tension on the chondrocyte's responsiveness to TGF-β3 and/or BMP7.

Purpose: A subset of estrogen receptor-positive (ER-positive) breast cancer (BC) contains high levels of tumor-infiltrating lymphocytes (TILs), similar to triple-negative BC (TNBC). The majority of immuno-oncology trials target TNBCs because of the greater proportion of TIL-rich TNBCs. The extent to which the immune microenvironments of immune-rich ER-positive BC and TNBC differ is unknown.

Patients and methods: RNA sequencing data from The Cancer Genome Atlas (TCGA; n = 697 ER-positive BCs; n = 191 TNBCs) were used for discovery; microarray expression data from Molecular Taxonomy of Breast Cancer International Consortium (METABRIC; n = 1,186 ER-positive BCs; n = 297 TNBCs) was used for validation. Patients in the top 25th percentile of a previously published total TIL metagene score distribution were considered immune rich. We compared expression of immune cell markers, immune function metagenes, and immuno-oncology therapeutic targets among immune-rich subtypes.

Results: Relative fractions of resting mast cells (TCGA P _adj = .009; METABRIC P _adj = 4.09E-15), CD8⁺ T cells (TCGA P _adj = .015; METABRIC P _adj = 0.390), and M2-like macrophages (TCGA P _adj= 4.68E-05; METABRIC P _adj = .435) were higher in immune-rich ER-positive BCs, but M0-like macrophages (TCGA P _adj = 0.015; METABRIC P _adj = .004) and M1-like macrophages (TCGA P _adj = 9.39E-08; METABRIC P _adj = 6.24E-11) were higher in immune-rich TNBCs. Ninety-one immune-related genes (eg, CXCL14, CSF3R, TGF-B3, LRRC32/GARP, TGFB-R2) and a transforming growth factor β (TGF-β) response metagene were significantly overexpressed in immune-rich ER-positive BCs, whereas 41 immune-related genes (eg, IFNG, PD-L1, CTLA4, MAGEA4) were overexpressed in immune-rich TNBCs in both discovery and validation data sets. TGF-β pathway member genes correlated negatively with expression of immune activation markers (IFNG, granzyme-B, perforin) and positively with M2-like macrophages (IL4, IL10, and MMP9) and regulatory T-cell (FOXP3) markers in both subtypes.

Conclusion: Different immunotherapy strategies may be optimal in immune-rich ER-positive BC and TNBC. Drugs targeting the TGF-β pathway and M2-like macrophages are promising strategies in immune-rich ER-positive BCs to augment antitumor immunity.

A key component of cardiac ischemia-reperfusion injury (IRI) is the increased generation of reactive oxygen species, leading to enhanced inflammation and tissue dysfunction in patients following intervention for myocardial infarction. In this study, we hypothesized that oxidative stress, due to ischemia-reperfusion, induces senescence which contributes to the pathophysiology of cardiac IRI. We demonstrate that IRI induces cellular senescence in both cardiomyocytes and interstitial cell populations and treatment with the senolytic drug navitoclax after ischemia-reperfusion improves left ventricular function, increases myocardial vascularization, and decreases scar size. SWATH-MS-based proteomics revealed that biological processes associated with fibrosis and inflammation that were increased following ischemia-reperfusion were attenuated upon senescent cell clearance. Furthermore, navitoclax treatment reduced the expression of pro-inflammatory, profibrotic, and anti-angiogenic cytokines, including interferon gamma-induced protein-10, TGF-β3, interleukin-11, interleukin-16, and fractalkine. Our study provides proof-of-concept evidence that cellular senescence contributes to impaired heart function and adverse remodeling following cardiac ischemia-reperfusion. We also establish that post-IRI the SASP plays a considerable role in the inflammatory response. Subsequently, senolytic treatment, at a clinically feasible time-point, attenuates multiple components of this response and improves clinically important parameters. Thus, cellular senescence represents a potential novel therapeutic avenue to improve patient outcomes following cardiac ischemia-reperfusion.

Keywords: cardiac; ischemia-reperfusion; remodeling; senescence; senolytic.