Tumor progression is governed by various growth factors and cytokines in the tumor microenvironment (TME). Among these, transforming growth factor-β (TGF-β) is secreted by various cell types residing in the TME and promotes tumor progression by inducing the epithelial-to-mesenchymal transition (EMT) of cancer cells and tumor angiogenesis. TGF-β comprises three isoforms, TGF-β1, -β2, and -β3, and transduces intracellular signals via TGF-β type I receptor (TβRI) and TGF-β type II receptor (TβRII). For the purpose of designing ligand traps that reduce oncogenic signaling in the TME, chimeric proteins comprising the ligand-interacting ectodomains of receptors fused with the Fc portion of immunoglobulin are often used. For example, chimeric soluble TβRII (TβRII-Fc) has been developed as an effective therapeutic strategy for targeting TGF-β ligands, but several lines of evidence indicate that TβRII-Fc more effectively traps TGF-β1 and TGF-β3 than TGF-β2, whose expression is elevated in multiple cancer types. In the present study, we developed a chimeric TGF-β receptor containing both TβRI and TβRII (TβRI-TβRII-Fc) and found that TβRI-TβRII-Fc trapped all TGF-β isoforms, leading to inhibition of both the TGF-β signal and of TGF-β-induced EMT of oral cancer cells, whereas TβRII-Fc failed to trap TGF-β2. Furthermore, we found that TβRI-TβRII-Fc suppresses tumor growth and angiogenesis more effectively than TβRII-Fc in a subcutaneous xenograft model of oral cancer cells with high TGF-β expression. These results suggest that TβRI-TβRII-Fc may be a promising tool for targeting all TGF-β isoforms in the TME.

Keywords: Fc receptor; angiogenesis; cancer therapy; epithelial-mesenchymal transition (EMT); ligand trap; oral squamous cell cancer; transforming growth factor beta (TGF-B); tumor microenvironment.

OBJECTIVE

We have recently shown that mesenchymal stem cells (MSCs) embedded in a hyaluronic acid (HA) hydrogel and exposed to chondrogenic factors (transforming growth factor-β3 [TGF-β3]) produce a cartilage-like tissue in vitro. The current objective was to determine if these same factors could be combined immediately prior to implantation to induce a superior healing response in vivo relative to the hydrogel alone.

METHODS

Trochlear chondral defects were created in Yucatan mini-pigs (6 months old). Treatment groups included an HA hydrogel alone and hydrogels containing allogeneic MSCs, TGF-β3, or both. Six weeks after surgery, micro-computed tomography was used to quantitatively assess defect fill and subchondral bone remodeling. The quality of cartilage repair was assessed using the ICRS-II histological scoring system and immunohistochemistry for type II collagen.

RESULTS

Treatment with TGF-β3 led to a marked increase in positive staining for collagen type II within defects (P < 0.05), while delivery of MSCs did not (P>> 0.05). Neither condition had an impact on other histological semiquantitative scores (P>> 0.05), and inclusion of MSCs led to significantly less defect fill (P < 0.05). For all measurements, no synergistic interaction was found between TGF-β3 and MSC treatment when they were delivered together (P>> 0.05).

CONCLUSIONS

At this early healing time point, treatment with TGF-β3 promoted the formation of collagen type II within the defect, while allogeneic MSCs had little benefit. Combination of TGF-β3 and MSCs at the time of surgery did not produce a synergistic effect. An in vitro precultured construct made of these components may be required to enhance in vivo repair in this model system.

Engineering the osteochondral tissue presents some challenges mainly relying in its function of transition from the subchondral bone to articular cartilage and the gradual variation in several biological, mechanical, and structural features. A possible solution for osteochondral regeneration might be the design and fabrication of scaffolds presenting a gradient able to mimic this transition. Covalent binding of biological factors proved to enhance cell adhesion and differentiation in two-dimensional culture substrates. Here, we used polymer brushes as selective linkers of bone morphogenetic protein-2 (BMP-2) and transforming growth factor-β3 (TGF-β3) on the surface of 3D scaffolds fabricated via additive manufacturing (AM) and subsequent controlled radical polymerization. These growth factors (GFs) are known to stimulate the differentiation of human mesenchymal stromal cells (hMSCs) toward the osteogenic and chondrogenic lineages, respectively. BMP-2 and TGF-β3 were covalently bound both homogeneously within a poly(ethylene glycol) (PEG)-based brush-functionalized scaffolds, and following a gradient composition by varying their concentration along the axial section of the 3D constructs. Following an approach previously developed by our group and proved to be successful to generate fibronectin gradients, opposite brush-supported gradients of BMP-2 and TGF-β3 were finally generated and subsequently tested to differentiate cells in a gradient fashion. The brush-supported GFs significantly influenced hMSCs osteochondral differentiation when the scaffolds were homogenously modified, yet no effect was observed in the gradient scaffolds. Therefore, this technique seems promising to maintain the biological activity of growth factors covalently linked to 3D scaffolds, but needs to be further optimized in case biological gradients are desired.

Hyaline articular cartilage, which protects the bones of diarthrodial joints from forces associated with load bearing, frictions, and impacts has very limited capacities for self-repair. Over the years, the trend of treatments has shifted to regenerations and researchers have been on the quest for a lasting regeneration. We evaluated the treatment of osteoarthritis by chondrogenically induced ADSCs and BMSCs for a long time functional recovery.

Osteoarthritis was induced at the right knee of sheep by complete resection of ACL and medial meniscus. Stem cells from sheep were induced to chondrogenic lineage. Test sheep received 5 mls single doses of 2 × 107 autologous PKH26-labelled ADSCs or BMSCs, while controls received basal medium. Functional recovery of the knees was evaluated via electromyography.

Induced ADSCs had 625, 255, 393, 908, 409, 157 and 1062 folds increases of collagen I, collagen II, aggrecan, SOX9, cartilage oligomeric protein, chondroadherin and fibromodullin compare to uninduced cells, while BMSCs had 702, 657, 321, 276, 337, 233 and 1163 respectively; p = .001. Immunocytochemistry was positive for these chondrogenic markers. 12 months post-treatment, controls scored 4 in most regions using ICRS, while the treated had 8; P = .001. Regenerated cartilages were positive to PKH26 and demonstrated the presence of condensing cartilages on haematoxylin and eosin; and Safranin O. OA degenerations caused significant amplitude shift from right to left hind limb. After treatments, controls persisted with significant decreases; while treated samples regained balance.

Both ADSCs and BMSCs had increased chondrogenic gene expressions using TGF-β3 and BMP-6. The treated knees had improved cartilage scores; PKH26 can provide elongated tracking, while EMG results revealed improved joint recoveries. These could be suitable therapies for osteoarthritis.

BACKGROUND

Chondrogenic differentiation of adipose-derived stem cells (ASCs) has proven to be feasible. To compensate for laryngeal palsy or cartilage defects after surgery or trauma using tissue engineering, a formable and stable scaffold material is mandatory.

METHODS

ASCs were seeded in fibrin-polyurethane scaffolds and cultured in chondrogenic differentiation medium adding the growth factors TGF-b1, TGF-b3, and BMP-2 for up to 35 days.

RESULTS

Histological examination showed acid glycosaminoglycans in the extracellular matrix in all groups. Immunofluorescence presented positive staining for collagen II, aggrecan, and SOX-9 in the TGF-b1-, TGF-b3-, and BMP-2-group. With Real-time PCR analyses, chondrogenic differentiation became apparent by the expression of the specific genes COL2A1 (collagen II), AGC 1 (aggrecan), and SOX-9, whereas collagen II expression was low in all groups compared to bone marrow-derived stem cells (BMSC) due to reduced chondrogenic ability.

CONCLUSIONS

These findings demonstrate the general ability of ASCs to differentiate into matrix-producing chondrocytes in fibrin-polyurethane scaffolds. However, further experiments are necessary to enhance this chondrogenic potential of ASCs seeded in fibrin-polyurethane scaffolds in order to produce a suitable regeneration method for treating cartilage defects or an implantable medialization material for vocal cord palsy.

In our quest to standardize our formula for a clinical trial, transforming growth factor-beta3 (TGF-β3) alone and in combination with bone morphogenetic protein-6 (BMP-6) were evaluated for their effectiveness in cartilage differentiation. Bone Marrow Stem Cells (BMSCs) and Adipose Derived Stem Cells (ADSCs) were induced to chondrogenic lineage using two different media. Native chondrocytes served as positive control. ADSCs and BMSCs proved multipotency by tri-lineage differentiations. ADSC has significantly higher growth kinetics compare to Chondrocyte only p ≤ 0.05. Using TGF-β3 alone, BMSC revealed higher expressions for hyaline cartilage genes compare to ADSCs. Chondrocyte has significantly higher early chondrogenic markers expression to ADSCs and BMSCs, while BMSCs was only higher to ADSC at chondroadherin, p ≤ 0.0001. On mature chondrogenic markers, chondrocytes were significantly higher to ADSCs and BMSCs for aggrecan, collagen IX, sry (sex determining region y)-box9, collagen II and fibromodullin; and only to ADSC for collagen XI. BMSC was higher to ADSC for aggrecan and collagen IX, p ≤ 0.0001. The combination of TGF-β3 + BMP-6 revealed increased gene expressions on both BMSCs and ADSCs for early and mature chondrogenic markers, but no significance difference. For dedifferentiation markers, ADSC was significantly higher to chondrocyte for collagen I. Glycosaminoglycan evaluations with both formulas revealed that chondrocytes were significantly higher to ADSCs and BMSCs, but none was significant to each other, p ≤ 0.0001. Combination of 10 ng TGF-β3 with 10 ng of BMP-6 enhanced chondrogenic potentials of BMSCs and ADSCs compare to TGF-β3 alone. This could be the ideal cocktail for either cell's chondrogenic induction.

To evaluate and compare transforming growth factor β3 (TGF-β3) serum concentration in patients with uterine fibroids (UFs) without hormone treatment, treated with ulipristal acetate (UPA), and controls; to evaluate TGF-β3 concentrations in UF tissue in patients without hormone treatment and those treated with UPA; and to evaluate the correlations of age and body mass index (BMI) with TGF-β3 serum and UF tissue levels between the groups.

Retrospective cohort study.

University teaching hospital.

A total of 141 patients divided into three groups: UFs non-UPA, UFs, and UPA, controls.

Medical history and examination, genital ultrasound scan, blood and tissue sampling, and measurement of TGF-β3 serum and tissue concentrations.

Evaluation of the impact of UPA (3 months treatment), age and BMI on TGF-β3 serum and UF tissue levels.

The values of TGF-β3 serum and tissue concentrations statistically significantly differed between the non-UPA and UPA groups. The mean TGF-β3 serum concentrations were non-UPA group 32.24 ± 34.55 pg/mL, UPA group 10.88 ± 7.15 pg/mL, and controls 11.97 ± 10.30 pg/mL. The mean TGF-β3 tissue concentrations were non-UPA group 171.29 ± 91.81 pg/mg and UPA group 99.99 ± 60.63 pg/mg. Statistically significantly lower mean TGF-β3 serum and tissue concentrations were observed in patients treated with UPA. No statistically significant correlations between TGF-β3 concentrations and age or BMI were found.

Reduction of serum and tissue TGF-β3 concentrations in UFs may be an important component of the effect of UPA on UF biology. Further research in this area is necessary.

The aim of this paper is to study the participation of transforming growth factor-β (TGF-β) signaling pathway in mediating the growth of human uterine leiomyoma (UL) activated by phenolic environmental estrogens (EEs), via the interaction between TGF-β and ER signaling pathways. The UL cells were prepared by primary culture and subculture methods. To validate the role of TGF-β3 (5 ng/ml) for the viability of human uterine leiomyoma cells, CCK-8 assay was performed in each of five treatment groups including E2 group (E2 10(9) mol/l), BPA group (bisphenol A 10 μmol/l), NP group (nonylphenol 32 μmol/l), OP group (octylphenol 8 μmol/l), or control group (DMSO only). Subsequently, qRT-PCR was applied to detect mRNA expressions of ERα and c-fos, while western blot assay was used to test the expressions of p-Smad3, SnoN, and c-fos proteins in all settings mentioned above; the expressions were compared among different groups, and also in settings with and without synchronous treatment of ICI 182,780. Primarily cultured UL cells were successfully established. Compared with the control group, there were statistically significant increases in the proliferation rate of the UL cells in all EE groups or treated with TGF-β3 only (p < 0.05). Nevertheless, a slight decrease in proliferation rate of UL was detected in coexistence with TGF-β3 in all EE groups (p>> 0.05). Interestingly, mRNA expressions of ERα and c-fos reduced in the setting of coexistence of TGF-β3 and EEs compared to isolated EE treatment (p < 0.05). Compared with the control group, the expression of p-Smad3 and c-fos proteins significantly decreased (p < 0.05) in each of E2, BPA, NP, and OP group, and the expression of SnoN protein also significantly reduced only in BPA and NP groups (p < 0.05), followed by TGF-β3 treatment. When adding ICI 182,780, the expression of p-Smad3 protein significantly increased in OP group (p < 0.05), but slightly increased in E2, BPA, NP, and OP groups (p>> 0.05). However, compared with the control group, the expressions of SnoN and c-fos proteins significantly decreased (p < 0.05) after adding ICI182,780. Moreover, there was a significant statistical difference in the expression of p-Smad3, SnoN, and c-fos proteins between pre- and post-treatment of ICI 182,780 in all groups (p < 0.05). The ERα signaling pathway and TGF-β signaling pathway have different roles in the control of UL cell proliferation. The phenolic EEs can be a promoter of UL cell proliferation, which is mediated by ERα signaling pathway and its cross-talking with TGF-β signaling pathway. Both less exposure to EEs and blockade of TGF signaling pathway are necessary strategies to prevent UL.

Bone marrow-derived mesenchymal stem cells (BMSCs) can be obtained by minimally invasive means and would be a favourable source for cell-based cartilage regeneration. However, controlling the differentiation of the BMSCs towards the desired chondrogenic pathway has been a challenge hampering their application. The major aim of the present study was to determine if conditioned medium collected from cultured auricular chondrocytes could promote chondrogenic differentiation of BMSCs. Auricular chondrocytes were isolated and grown in BMSC standard culture medium (SM) that was collected and used as chondrocyte-conditioned medium (CCM). The BMSCs were expanded in either CCM or SM for three passages. Cells were seeded onto fibrous collagen scaffolds and precultured for 2 weeks with or without transforming growth factor-beta 3 (TGF-β3). After preculture, constructs were implanted subcutaneously in nude mice for 6 and 12 weeks and evaluated with real-time polymerase chain reaction, histology, immunohistochemistry and biochemistry. Real-time polymerase chain reaction results showed upregulation of COL2A1 in the constructs cultured in CCM compared with those in SM. After 12 weeks in vivo, abundant neocartilage formation was observed in the implants that had been cultured in CCM, with or without TGF-β3. In contrast, very little cartilage matrix formation was observed within the SM groups, regardless of the presence of TGF-β3. Osteogenesis was only observed in the SM group with TGF-β3. In conclusion, CCM even had a stronger influence on chondrogenesis than the supplementation of the standard culture medium with TGF-β3, without signs of endochondral ossification. Efficient chondrogenic differentiation of BMSCs could provide a promising alternative cell population for auricular regeneration. Copyright © 2016 John Wiley & Sons, Ltd.

Well documented limitations associated with primary chondrocytes for cartilage tissue engineering applications have led to increased interest in the use of multi-potent stem/progenitor cells. The objective of this study was to firstly investigate if infrapatellar fat pad-derived stem cells (FPSCs) could be used to engineer cartilage-like tissues through a self-assembly (SA) process, and secondly to compare the properties of such grafts to those engineered by agarose hydrogel encapsulation (AE). Self-assembled cartilaginous tissues were first engineered by geometrically confining FPSCs on tissue culture plastic, and then either continuously or transiently supplementing these constructs with transforming growth factor-b3 (TGF-b3). Transient supplementation with TGF-b3 (for the first 21 days of culture) enhanced the development of self-assembled grafts, with sGAG accumulation reaching levels of 8.4 ± 1.5% w/w after 6 weeks of culture. While overall levels of matrix synthesis were higher with AE compared to SA, when normalized to tissue wet weight, ECM accumulation was significantly greater in the lighter SA constructs. A potential drawback with the SA approach on tissue culture plastic was that it often led to the development of contracted,geometrically inconsistent tissues.We therefore next explored if SA on polyethylene terephthalate (PET) transwell membranes would lead to the development of more morphologically stable and homogenous tissues. At high seeding densities, SA on such transwell membranes led to the formation of geometrically uniform constructs that underwent minimal contraction during culture. In conclusion, the results of this study demonstrate the potential of SA using FPSCs for cartilage tissue engineering, with grafts attaining relatively high levels of sGAG content within clinically relevant timeframes. Such an approach is easily scalable and may lend itself to treating large, full thickness cartilage defects.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by progressive muscle paralysis. Respiratory complications are the main cause of death in ALS. For this reason, initial respiratory status and its decline over disease progression are strong independent predictors of survival. Riluzole, a glutamatergic neurotransmission inhibitor, is the only drug that has shown to extend survival. Therefore, both novel molecular biomarkers and treatment strategies are needed. Transforming growth factor-β (TGF-β) family cytokines are important regulators of cell fate affecting both neurogenesis and neurodegeneration. Several studies demonstrate that TGF-β signalling protects neurons from glutamate-mediated excitotoxicity, a recognized mechanism underlying the pathogenesis of various neurodegenerative disorders such as ALS. Recent studies report dysregulations of the TGF-β system as a common feature of neurodegenerative disorders. The upregulation of this system has been linked with ALS progression. We have quantified TGF-β1, TGF-β2 and TGF-β3 serum levels in 23 ALS patients and 12 healthy controls, our preliminary results support the hypothesis that TGF-β3 levels can be a marker disease severity ALS. Further results are necessary to confirm this hypothesis.

Osteoarthritis (OA) is the most common chronic joint disease and leads to the degradation of the extracellular matrix by an imbalance between anabolic and catabolic processes. TGF-β3 (transforming growth factor beta-3) and epidermal growth factor (EGF) influence the osteochondrogenic potential of chondrocytes. In this study, we compared the expression of mediators and receptors in the TGF-β3 and EGF pathways, as well as biglycan (BGN), in healthy and diseased chondrocytes. Furthermore, we used chondrogenic progenitor cells (CPCs) for in vitro stimulation and knockdown experiments to elucidate the effects of TGF-β3 and EGF on the chondrogenic potential. Our results demonstrate that the expression of TGF-beta receptor type-1 (TGFBRI) and epidermal growth factor receptor (EGFR) is altered in diseased chondrocytes as well as in CPCs. Moreover, TGF-β3 and EGF stimulation influenced the expression levels of BGN, SRY (sex determining region Y)-box 9 (SOX9), and Runt-related transcription factor 2 (RUNX2) in CPCs. Therefore, changes in TGFBRI and EGFR expression likely contribute to the degenerative and regenerative effects seen in late stages of OA.

Intervertebral disc (IVD) repair is a high-priority topic in our active and increasingly ageing society. Since a high number of people are affected by low back pain treatment options that are able to restore the biological function of the IVD are highly warranted. Here, we investigated whether the feasibility of genetically engineered (GE)-silk from Bombyx mori containing specific growth factors to precondition human bone-marrow derived mesenchymal stem cells (hMSC) or to activate differentiated human annulus fibrosus cells (hAFC) prior transplantation or for direct repair on the IVD. Here, we tested the hypothesis that GE-silk fleece can thrive human hMSC towards an IVD-like phenotype. We aimed to demonstrate a possible translational application of good manufacturing practice (GMP)-compliant GE-silk scaffolds in IVD repair and regeneration. GE-silk with growth and differentiation factor 6 (GDF-6-silk) or transforming growth factor β3 (TGF-β3, TGF-β3-silk) and untreated silk (cSilk) were investigated by DNA content, cell activity assay and glycosaminoglycan (GAG) content and their differentiation potential by qPCR analysis. We found that all silk types demonstrated a very high biocompatibility for both cell types, that is, hMSC and hAFC, as revealed by cell activity, and DNA proliferation assay. Further, analyzing qPCR of marker genes revealed a trend to differentiation toward an NP-like phenotype looking at the Aggrecan/Collagen 2 ratio which was around 10:1. Our results support the conclusion that our GE-silk scaffold treatment approach can thrive hMSC towards a more IVD-like phenotype or can maintain the phenotype of native hAFC. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:1324-1333, 2018.

Numerous growth factor delivery systems have been developed for tissue engineering. However, little is known about how the dose of a specific protein will influence tissue regeneration, or how different patients will respond to altered levels of growth factor presentation. The objective of the present study was to assess stem cell chondrogenesis within extracellular-matrix (ECM)-derived scaffolds loaded with escalating levels of transforming growth factor (TGF)-β3. It was also sought to determine if stem cells display a donor-dependent response to different doses of TGF-β3, from low (5 ng) to high (200 ng), released from such scaffolds. It was found that ECM-derived scaffolds possess the capacity to bind and release increasing amounts of TGF-β3, with between 60% and 75% of this growth factor released into the media over the first 12 days of culture. After seeding these scaffolds with human infrapatellar fat pad-derived stem cells (FPSCs), it was found that cartilage-specific ECM accumulation was greatest for the higher levels of growth factor loading. Importantly, soak-loading cartilage ECM-derived scaffolds with high levels of TGF-β3 always resulted in at least comparable levels of chondrogenesis to controls where this growth factor was continuously added to the culture media. Similar results were observed for FPSCs from all donors, although the absolute level of secreted matrix did vary from donor to donor. Therefore, while no single growth factor release profile will be optimal for all patients, the results of this study suggest that the combination of a highly porous cartilage ECM-derived scaffold coupled with appropriate levels of TGF-β3 can consistently drive chondrogenesis of adult stem cells. Copyright © 2016 John Wiley & Sons, Ltd.

Although caffeine and glucocorticoids are frequently used to treat chronic lung disease in preterm neonates, potential interactions are largely unknown. While anti-inflammatory effects of glucocorticoids are well defined, their impact on airway remodeling is less characterized. Caffeine has been ascribed to positive effects on airway inflammation as well as remodeling. Connective tissue growth factor (CTGF, CCN2) plays a key role in airway remodeling and has been implicated in the pathogenesis of chronic lung diseases such as bronchopulmonary dysplasia (BPD) in preterm infants. The current study addressed the impact of glucocorticoids on the regulation of CTGF in the presence of caffeine using human lung epithelial and fibroblast cells.

The human airway epithelial cell line H441 and the fetal lung fibroblast strain IMR-90 were exposed to different glucocorticoids (dexamethasone, budesonide, betamethasone, prednisolone, hydrocortisone) and caffeine. mRNA and protein expression of CTGF, TGF-β1-3, and TNF-α were determined by means of quantitative real-time PCR and immunoblotting. H441 cells were additionally treated with cAMP, the adenylyl cyclase activator forskolin, and the selective phosphodiesterase (PDE)-4 inhibitor cilomilast to mimic caffeine-mediated PDE inhibition.

Treatment with different glucocorticoids (1 μM) significantly increased CTGF mRNA levels in H441 (p < 0.0001) and IMR-90 cells (p < 0.01). Upon simultaneous exposure to caffeine (10 mM), both glucocorticoid-induced mRNA and protein expression were significantly reduced in IMR-90 cells (p < 0.0001). Of note, 24 h exposure to caffeine alone significantly suppressed basal expression of CTGF mRNA and protein in IMR-90 cells. Caffeine-induced reduction of CTGF mRNA expression seemed to be independent of cAMP levels, adenylyl cyclase activation, or PDE-4 inhibition. While dexamethasone or caffeine treatment did not affect TGF-β1 mRNA in H441 cells, increased expression of TGF-β2 and TGF-β3 mRNA was detected upon exposure to dexamethasone or dexamethasone and caffeine, respectively. Moreover, caffeine increased TNF-α mRNA in H441 cells (6.5 ± 2.2-fold, p < 0.05) which has been described as potent inhibitor of CTGF expression.

In addition to well-known anti-inflammatory features, glucocorticoids may have adverse effects on long-term remodeling by TGF-β1-independent induction of CTGF in lung cells. Simultaneous treatment with caffeine may attenuate glucocorticoid-induced expression of CTGF, thereby promoting restoration of lung homeostasis.

Cadmium (Cd) impairs blood-testis barrier (BTB). Polydeoxyribonucleotide (PDRN), an adenosine A2A agonist, has positive effects on male reproductive system. We investigated the effects of PDRN on the morphological and functional changes induced by Cd in mice testes. Adult Swiss mice were divided into four groups: controls administered with 0.9% NaCl (1 ml/kg, i.p., daily) or with PDRN (8 mg/kg, i.p. daily), animals challenged with Cd chloride (CdCl2; 2 mg/kg, i.p, daily) and animals challenged with CdCl2 (2 mg/kg, i.p., daily) and treated with PDRN (8 mg/kg, i.p., daily). Experiments lasted 14 days. Testes were processed for biochemical, structural, and ultrastructural evaluation and hormones were assayed in serum. CdCl2 increased pERK 1/2 expression and Follicle Stimulating Hormone (FSH) and Luteinizing Hormone (LH) levels; it decreased testosterone (TE) and inhibin-B levels and induced structural damages in extratubular compartment and in seminiferous epithelium, with ultrastructural features of BTB disruption. Many TUNEL-positive germ cells were present. CdCl2 increased tubular TGF-β3 immunoreactivity and reduced claudin-11, occludin, and N-cadherin immunoreactivity. PDRN administration reduced pERK 1/2 expression, FSH, and LH levels; it increased TE and inhibin-B levels, ameliorated germinal epithelium changes and protected BTB ultrastructure. Few TUNEL-positive germ cells were present and the extratubular compartment was preserved. Furthermore, PDRN decreased TGF-β3 immunoreactivity and enhanced claudin-11, occludin, and N-cadherin immunoreactivity. We demonstrate a protective effect of PDRN on Cd-induced damages of BTB and suggest that PDRN may play an important role against Cd, particularly against its harmful effects on gametogenesis.

Nitric oxide (NO) is an important bioactive molecule with regulatory, cytotoxic or cytoprotective properties. In virus-induced myocarditis, NO mediates host defense mechanisms against the infection or causes cardiac dysfunctions. NO is synthesized from L-arginine by the enzyme nitric oxide synthase (NOS). The expression of the inducible form of the nitric oxide synthase (iNOS) is regulated by cytokines, involved in the complex myocardial immune response to enterovirus infections. The present study was undertaken to characterize the role of iNOS and NO in the murine model of viral myocarditis induced by coxsackievirus B3 (CVB3). In response to CVB3 infection we investigated the time course of iNOS induction in correlation with cytokine mRNA expression (TNF-alpha, IL-1 alpha, IFN-gamma, TGF-beta) in the heart of NMRI mice by RT-PCR. Positive PCR signals for viral RNA were found in the acute and chronic stage of disease by seminested PCR, indicating the persistence of viral genome. We found distinct expression of iNOS at all time points (1, 2, 3, 4, 7, 14, 28, 56, 98 days post infection [p.i.]). Higher iNOS mRNA levels were identified between days 4 until 28 p.i. in comparison to day 56 and 98 p.i. using densitometric values. The mRNA of the inflammatory cytokines TNF-alpha, IL-1 alpha, IFN-gamma appeared at days 1, 4, and 7 p.i., peaked at day 7 p.i. and persisted until day 98 p.i. Similar like the iNOS mRNA pattern was the expression profile of TGF-beta. Using in situ hybridization and immunohistochemistry iNOS was localized in infiltrates, vascular endothelial cells, smooth muscle cells, myocytes and throughout the interstitial spaces between myocardial fibers in the heart sections of NMRI mice. Increased levels of NO were measured as total nitrate/nitrite concentration in the sera of mice from day 7 until day 28 p.i.

The present study aimed to investigate the protective effect of cultured Cordyceps sinensis polysaccharides (CSP) on cyclophosphamide (Cy)-induced intestinal mucosal immunosuppression and microbial dysbiosis in mice. Results showed that CSP stimulated cytokines secretion (IL-12, IFN-γ, IL-4, IL-13, IL-6, IL-17, IL-10, TGF-β3, TNF-α, IL-2, IL-21) and transcription factors production (T-bet, GATA-3, RORγt, Foxp3). TLRs (TLR-2, TLR-4, TLR-6) and NF-κB pathway key proteins (p-IκB-α, NF-κB p65) were also upregulated after CSP administration. Moreover, CSP recovered SCFAs levels which decreased by Cy treatment. Furthermore, 16S rRNA sequencing of fecal samples was performed. α-diversity and β-diversity analysis revealed CSP improved microbial community diversity and modulated the overall structure of gut microbiota. Taxonomic composition analysis found that CSP increased the abundance of probiotics (Lactobacillus, Bifidobacterium, Bacteroides) and decreased pathogenic bacteria (Clostridium, Flexispira). These findings suggested the potential of CSP as a prebiotics to reduce side effects of Cy on intestinal mucosal immunity and gut microbiota.

Autologous chondrocytes remain one of the most preferable candidates among various therapeutic cell species because of their high efficacy, despite remarkable progress in discovery and development of therapeutic cells for cartilage regenerative medicine to date. However, the essential process of cell expansion via repeated monolayer sub-cultures inevitably induces chondrocytic dedifferentiation. In this study, we aimed to achieve and enhance redifferentiation of dedifferentiated chondrocytes with dual genes of transforming growth factor (TGF)-β3 and short hairpin RNA (shRNA) that restore chondrocytic phenotype and silence fibrous collagen type I (Col I), respectively. It was hypothesized that gene delivery of the two targets would promote chondrogenesis in chondrocytes, and meanwhile inhibit the expression of the undesired Col I. Three types of recombinant adenoviruses were constructed. Two of them were of single-function vectors with the ability to express either TGF-β3 (Ad-TGFβ3) or shRNA (specific for Col I, Ad-shRNA); the third type was of double-function vectors that encode both TGF-β3 and anti-Col I shRNA (Ad-double). We infected the dedifferentiated chondrocytes with Ad-double, or co-transduced them with Ad-TGFβ3 and Ad-shRNA at the same time (designated as Ad-combination). Data from real-time RT-PCR and histological staining suggested a restoration in the expression of cartilage-specific genes including aggrecan, type II collagen, and cartilage oligomeric matrix protein (COMP); while a significant down-regulation of Col I expression was observed in groups treated with Ad-double and Ad-combination compared to other control groups. These results demonstrated that, by genetic modification, dedifferentiated chondrocytes managed to redifferentiate back to chondrocytic phenotype, which may greatly facilitate cartilage regenerative medicine by providing sufficient number of competent therapeutic cells.

To regenerate articular cartilage tissue from degeneration and trauma, synovial mesenchymal stem cells (SMSCs) were used in this study as therapeutic progenitor cells to induce therapeutic chondrogenesis. To accomplish this, chondrocytes pre-transduced with adenoviral vectors carrying the transforming growth factor (TGF) β3 gene were selected as transgenic companion cells and co-cultured side-by-side with SMSCs in a 3D environment to provide chondrogenic growth factors in situ. We adopted a mixed co-culture strategy for this purpose. Transgenic delivery of TGF-β3 in chondrocytes was performed via recombinant adenoviral vectors. The mixed co-culture of SMSCs and transgenic chondrocytes was produced in alginate gel constructs. Gene expression in both SMSCs and chondrocytes were characterized. Biochemical assays in vitro and in vivo showed that release of TGF-ß3 from transgenic chondrocytes not only induced SMSC differentiation into chondrocytic cells but also preserved the chondrocytic phenotype of chondrocytes from suspected dedifferentiation. As a result, this mixed co-culture strategy in conjunction with TGF-ß3 gene delivery could be a promising approach in cartilage tissue engineering.

Despite extensive efforts to develop efficacious therapeutic approaches, the treatment of skin wounds remains a considerable clinical challenge. Existing remedies cannot sufficiently meet current needs, so the discovery of novel pro-healing agents is of growing importance. In the current research, we identified a novel short peptide (named RL-QN15, primary sequence 'QNSYADLWCQFHYMC') from Rana limnocharis skin secretions, which accelerated wound healing in mice. Exploration of the underlying mechanisms showed that RL-QN15 activated the MAPK and Smad signaling pathways, and selectively modulated the secretion of cytokines from macrophages. This resulted in the proliferation and migration of skin cells and dynamic regulation of TGF-β1 and TGF-β3 in wounds, which accelerated re-epithelialization and granulation tissue formation and thus skin regeneration. Moreover, RL-QN15 showed significant therapeutic potency against chronic wounds, skin fibrosis, and oral ulcers. Our results highlight frog skin secretions as a potential treasure trove of bioactive peptides with healing activity. The novel peptide (RL-QN15) identified in this research shows considerable capacity as a candidate for the development of novel pro-healing agents.

Keywords: Acetic Acid: (PubChem CID: 176); Bleomycin: (PubChem CID: 5360373); Exendin-4 (PubChem CID: 45588096); Lipopolysaccharide (PubChem CID: 75107052); Pentobarbital sodium: (PubChem CID: 23676152); RL-QN15; Streptozotocin (PubChem CID: 29327); Wound healing; amphibian; peptide; pro-healing agents.

Transforming growth factor-beta3 (TGF-β3) and 1α,25-dihydroxyvitamin D3 (1α,25 (OH) 2D3) are essential factors in chondrogenesis and osteogenesis respectively. These factors also play a fundamental role in the developmental processes and the maintenance of skeletal integrity, but their respective direct effects on these processes are not fully understood. Using an organotypic bone rudiment culture system the current study has examined the direct roles the osteotropic factors 1α,25 (OH)2D3 and TGF-β3 exert on the development and modulation of the three dimensional structure of the embryonic femur. Isolated embryonic chick femurs (E11) were organotypically cultured for 10 days in basal media, or basal media supplemented with either 1α,25 (OH) 2D3 (25 nM) or TGF-β3 (5 ng/mL & 15 ng/mL). Analyses of the femurs were undertaken using micro-computed tomography (μCT), histology and immunohistochemistry. 1α,25 (OH)2D3 supplemented cultures enhanced osteogenesis directly in the developing femurs with elevated levels of osteogenic markers such as type 1 collagen. In marked contrast organotypic femur cultures supplemented with TGF-β3 (5 ng/mL & 15 ng/mL) demonstrated enhanced chondrogenesis with a reduction in osteogenesis. These studies demonstrate the efficacy of the ex vivo organotypic embryonic femur culture employed to elucidate the direct roles of these molecules, 1α,25 (OH) 2D3 and TGF-β3 on the structural development of embryonic bone within a three dimensional framework. We conclude that 1α,25(OH)2D and TGF-β3 modify directly the various cell populations in bone rudiment organotypic cultures effecting tissue metabolism resulting in significant changes in embryonic bone growth and modulation. Understanding the roles of osteotropic agents in the process of skeletal development is integral to developing new strategies for the recapitulation of bone tissue in later life.

Morinda officinalis is a traditional Chinese herbal medicine, which has been used to tonify the kidney and strengthen yang for a long time in China. In this study, the effects of M. officinalis Polysaccharide (MOP) on experimental varicocele adolescent rats were investigated. The result showed that varicocele destroyed the structure of the seminiferous epithelium and decreased the TJ protein expression (Occludin, Claudin-11, and ZO-1), testosterone (T) concentration in the left testicular tissue and serum, and serum levels of inhibin B (INHB), while increasing the levels of cytokines (TGF-β3 and TNF-α) in the left testicular tissue, as well as serum levels of gonadotropin-releasing hormone (GnRH), follicle-stimulating hormone (FSH), luteinizing hormone (LH), and antisperm antibody (AsAb). MOP repaired the damaged seminiferous epithelium and TJ and reduced the levels of cytokines (TGF-β3 and TNF-α) as well as serum levels of GnRH, FSH, LH, and AsAb, while upregulating TJ protein expression, T level in the left testicular tissue and serum, and serum INHB levels. In summary, we conclude that MOP promotes spermatogenesis and counteracts the varicocele-induced damage to the seminiferous epithelium and TJ, probably via decreasing cytokines (TGF-β3 and TNF-α) levels and regulating the abnormal sex hormones levels in experimental varicocele rats.

In order to explore the possibility of l-carnitine (LC) as a protector of male fertility in chemotherapy, we observed the damage of cyclophosphamide (CTX) to Sertoli cells and the protective effect of LC on the testis Sertoli cells from such damage in this study. Healthy adult male mice were divided into three groups: chemotherapy group were injected intraperitoneally with the CTX; protective agent group were injected both LC and CTX; control group mice were injected only with isochoric physiological saline; all once a day for 5 days. After 5 days, the mice were, respectively, killed at 24 hr after the last injection. The testis and epididymis were removed. Epididymis was for sperm analysis immediately, and immunohistochemistry, RT-PCR and Western blot for the assessments of occludin, glial cell-derived neurotrophic factor (GDNF) and TGF-β3 mRNA and protein expression. The sperm analysis of epididymis showed that CTX can significantly decrease sperm count and motility; and administration of LC resulted in significant recovery of the sperm count and sperm motility. Compared with control group, the expressions of occludin and GDNF decreased and the expression of TGF-β3 increased significantly (p < 0.05) in the CTX group. In the LC + CTX group, the expressions of occludin and GDNF were higher than those of the CTX group and similar to those of the control group; the TGF-β3 expression was lower (p < 0.05) than that of the CTX group and similar to that of the control group. The results of this study showed that CTX could damage the spermatogenesis and reduce the expression of occludin and GDNF, and increase the expression of TGF-β3 in testis of mouse, which indicates CTX's damage or efficacy to testis Sertoli cells. LC could protect the Sertoli cells of testis from these damages caused by CTX, and promote or protect the spermatogenesis. In conclusion, this study provides meaningful information about the possible damage to male fertility by chemotherapeutics and potential of LC in the protection of male fertility during chemotherapy.