Chronic airway remodeling is a serious consequence of asthma, which is caused by complex but largely unknown mechanisms. Despite versatile functions, the role of Lyn in chronic airway remodeling remains undefined. Using Lyn(-/-) mice, we show that continual exposure (for 8 wk) of house dust mite extracts induced a severe phenotype of chronic airway remodeling, including exacerbated mucus production, collagen deposition, dysregulated cytokine secretion, and elevated inflammation. Strikingly, a significant increase in TGF-β3 rather than TGF-β1 was observed in Lyn(-/-) mouse lungs compared with lungs in wild-type mice. Furthermore, TGF-β3 neutralizing Abs not only inhibited the expression of STAT6 and Smad2/3 but also decreased phosphorylation of Smad2 and NF-κB in Lyn(-/-) mouse lungs. In addition, both recombinant and adenoviral TGF-β3 significantly promoted epithelial-to-mesenchymal transition and intensified collagen I production and MUC5AC expression. Further examination of chronic asthma patients showed that a decreased Lyn correlated with the severity of airway inflammation and mucus hypersecretion. Finally, Lyn may critically regulate airway remodeling by directly interacting with TGF-β3. Collectively, these findings revealed that Lyn regulates TGF-β3 isoform and modulates the development of airway remodeling, which may have therapeutic implications for severe chronic asthma.

Epithelial-to-mesenchymal transition (EMT) plays a critical role in cancer metastasis, and is regulated by growth factors such as transforming growth factor β (TGF-β) and fibroblast growth factors (FGF) secreted from the stromal and tumor cells. However, the role of growth factors in EMT has not been fully established. Several integrins are upregulated by TGF-β1 during EMT. Integrins are involved in growth factor signaling through integrin-growth factor receptor crosstalk. We previously reported that FGF1 directly binds to integrin αvβ3 and the interaction was required for FGF1 functions such as cell proliferation and migration. We studied the role of αvβ3 induced by TGF-β on TGF-β-induced EMT. Here, we describe that FGF1 augmented EMT induced by TGF-β1 in MCF10A and MCF12A mammary epithelial cells. TGF-β1 markedly amplified integrin αvβ3 and FGFR1 (but not FGFR2). We studied if the enhancing effect of FGF1 on TGF-β1-induced EMT requires enhanced levels of both integrin αvβ3 expression and FGFR1. Knockdown of β3 suppressed the enhancement by FGF1 of TGF-β1-induced EMT in MCF10A cells. Antagonists to FGFR suppressed the enhancing effect of FGF1 on EMT. Integrin-binding defective FGF1 mutant did not augment TGF-β1-induced EMT in MCF10A cells. These findings suggest that enhanced integrin αvβ3 expression in addition to enhanced FGFR1 expression is critical for FGF1 to augment TGF-β1-induced EMT in mammary epithelial cells.

OBJECTIVE

Mesenchymal stem cells (MSCs) can differentiate into cells of mesenchymal lineages, such as osteoblasts and chondrocytes. Here we investigated the effects of IL-17, a key cytokine in chronic inflammation, on chondrogenic differentiation of human MSCs.

METHODS

Human bone marrow MSCs were pellet cultured in chondrogenic induction medium containing TGF-β3. Chondrogenic differentiation was detected by cartilage matrix accumulation and chondrogenic marker gene expression.

RESULTS

Over-expression of cartilage matrix and chondrogenic marker genes was noted in chondrogenic cultures, but was inhibited by IL-17 in a dose-dependent manner. Expression and phosphorylation of SOX9, the master transcription factor for chondrogenesis, were induced within 2 days and phosphorylated SOX9 was stably maintained until day 21. IL-17 did not alter total SOX9 expression, but significantly suppressed SOX9 phosphorylation in a dose-dependent manner. At day 7, IL-17 also suppressed the activity of cAMP-dependent protein kinase A (PKA), which is known to phosphorylate SOX9. H89, a selective PKA inhibitor, also suppressed SOX9 phosphorylation, expression of chondrogenic markers and cartilage matrix, and also decreased chondrogenesis.

CONCLUSIONS

IL-17 inhibited chondrogenesis of human MSCs through the suppression of PKA activity and SOX9 phosphorylation. These results suggest that chondrogenic differentiation of MSCs can be inhibited by a mechanism triggered by IL-17 under chronic inflammation.

OBJECTIVE

We have previously shown that TGF-β3 (T3) stimulates extracellular matrix (ECM) assembly while maintaining antifibrotic characteristics in a model using human corneal fibroblasts (HCFs). This model, however, requires non-physiological levels of serum. In the current study, we tested whether T3 could stimulate human corneal keratocytes (HCKs) in vitro to assemble a functional ECM, while maintaining their characteristics.

METHODS

Human corneal keratocytes and HCFs were isolated and cultured using 1% or 10% serum, respectively ±T3. The constructs were processed for indirect immunofluorescence (IF), transmission electron microscopy (TEM), and qRT-PCR, analyzing for keratocyte marker, keratocan, and ECM components, collagen (col) types I, III, and V.

RESULTS

Quantitative reverse transcriptase PCR data showed that keratocan, col I, and V were all upregulated in HCKs compared with HCFs, whereas col III was expressed at low levels in HCKs. Transforming growth factor beta 3 stimulation further enhanced the level of change. Without T3, HCK constructs were very thin, approximately 5 μm; however, as with HCFs, upon stimulation with T3, HCK constructs increased in thickness by approximately 5-fold. Cell counts and ECM production revealed that HCKs assembled more ECM per unit area compared with HCFs, and IF revealed downregulation of fibrotic markers, col III, and thrombospondin-1, with T3 stimulation. Transmission electron microscopy data revealed aligned ECM with long fibrils for all conditions except HCK Controls. Human corneal keratocytes+T3 also showed denser collagen fibrils with more consistent fibril diameter.

CONCLUSIONS

Overall, the data suggests that it is possible to stimulate matrix secretion and assembly by HCKs in vitro by using a single growth factor, T3.

The emergence of Th17 cells as a unique sub-population of CD4(+) T cells has revolutionized the current understanding of adaptive immune system and autoimmune diseases. Th17 cells are characterized by the expression of effector cytokines IL-17A, IL-17F, IL-21 and IL-22, and lineage specific transcription factor ROR-C in human and ROR-γt in mice. Generation and differentiation of Th17 cells from naive CD4(+) T cells is driven by transforming growth factor (TGF)-β, IL-6, IL-23, IL-1β and IL-21. Recent studies suggest that the pathogenicity of Th17 cells is determined by the presence of IL-23 and TGF-β3 in local micro-environment. Emerging reports highlight the importance of T-helper cell plasticity in pathogenesis of various autoimmune diseases. Th17 cells exhibit significant plasticity and converted to Th1-like cells under pathogenic conditions. Albeit growing body of evidences stating a pathogenic role for Th17 cells in autoimmune diabetes, conflicting reports also state an indifferent or protective role for Th17 cells. The operating mechanisms modulating Th17 immune response in autoimmune diabetes remain elusive. This review discusses recent advances in the understanding of transcriptional and post-transcriptional mechanisms of Th17 polarization, factors influencing pathogenicity of Th17 cells, molecular mechanisms of Th17/Th1 and Treg/Th17 plasticity and implications of these phenomena in autoimmune diabetes.

The regeneration of complex tissues and organs remains a major clinical challenge. With a view towards bioprinting such tissues, we developed a new class of pore-forming bioink to spatially and temporally control the presentation of therapeutic genes within bioprinted tissues. By blending sacrificial and stable hydrogels, we were able to produce bioinks whose porosity increased with time following printing. When combined with amphipathic peptide-based plasmid DNA delivery, these bioinks supported enhanced non-viral gene transfer to stem cells in vitro. By modulating the porosity of these bioinks, it was possible to direct either rapid and transient (pore-forming bioinks), or slower and more sustained (solid bioinks) transfection of host or transplanted cells in vivo. To demonstrate the utility of these bioinks for the bioprinting of spatially complex tissues, they were next used to zonally position stem cells and plasmids encoding for either osteogenic (BMP2) or chondrogenic (combination of TGF-β3, BMP2 and SOX9) genes within networks of 3D printed thermoplastic fibers to produce mechanically reinforced, gene activated constructs. In vivo, these bioprinted tissues supported the development of a vascularised, bony tissue overlaid by a layer of stable cartilage. When combined with multiple-tool biofabrication strategies, these gene activated bioinks can enable the bioprinting of a wide range of spatially complex tissues.

This study investigated the ability of chondrogenic and osteogenic predifferentiation of mesenchymal stem cells (MSCs) to play a role in the development of osteochondral tissue constructs using injectable bilayered oligo(poly(ethylene glycol) fumarate) (OPF) hydrogel composites. We hypothesized that the combinatorial approach of encapsulating cell populations of both chondrogenic and osteogenic lineages in a spatially controlled manner within bilayered constructs would enable these cells to maintain their respective phenotypes via the exchange of biochemical factors even without the influence of external growth factors. During monolayer expansion prior to hydrogel encapsulation, it was found that 7 (CG7) and 14 (CG14) days of MSC exposure to TGF-β3 allowed for the generation of distinct cell populations with corresponding chondrogenic maturities as indicated by increasing aggrecan and type II collagen/type I collagen expression. Chondrogenic and osteogenic cells were then encapsulated within their respective (chondral/subchondral) layers in bilayered hydrogel composites to include four experimental groups. Encapsulated CG7 cells within the chondral layer exhibited enhanced chondrogenic phenotype when compared to other cell populations based on stronger type II collagen and aggrecan gene expression and higher glycosaminoglycan-to-hydroxyproline ratios. Osteogenic cells that were co-cultured with chondrogenic cells (in the chondral layer) showed higher cellularity over time, suggesting that chondrogenic cells stimulated the proliferation of osteogenic cells. Groups with osteogenic cells displayed mineralization in the subchondral layer, confirming the effect of osteogenic predifferentiation. In summary, it was found that MSCs that underwent 7 days, but not 14 days, of chondrogenic predifferentiation most closely resembled the phenotype of native hyaline cartilage when combined with osteogenic cells in a bilayered OPF hydrogel composite, indicating that the duration of chondrogenic preconditioning is an important factor to control. Furthermore, the respective chondrogenic and osteogenic phenotypes were maintained for 28 days in vitro without the need for external growth factors, demonstrating the exciting potential of this novel strategy for the generation of osteochondral tissue constructs for cartilage engineering applications.

The objective of this study was to develop a scaffold derived from cartilaginous extracellular matrix (ECM) that could be used as a growth factor delivery system to promote chondrogenesis of stem cells. Dehydrothermal crosslinked scaffolds were fabricated using a slurry of homogenized porcine articular cartilage, which was then seeded with human infrapatellar-fat-pad-derived stem cells (FPSCs). It was found that these ECM-derived scaffolds promoted superior chondrogenesis of FPSCs when the constructs were additionally stimulated with transforming growth factor (TGF)-β3. Cell-mediated contraction of the scaffold was observed, which could be limited by the additional use of 1-ethyl-3-3dimethyl aminopropyl carbodiimide (EDAC) crosslinking without suppressing cartilage-specific matrix accumulation within the construct. To further validate the utility of the ECM-derived scaffold, we next compared its chondro-permissive properties to a biomimetic collagen-hyaluronic acid (HA) scaffold optimized for cartilage tissue engineering (TE) applications. The cartilage-ECM-derived scaffold supported at least comparable chondrogenesis to the collagen-HA scaffold, underwent less contraction and retained a greater proportion of synthesized sulfated glycosaminoglycans. Having developed a promising scaffold for TE, with superior chondrogenesis observed in the presence of exogenously supplied TGF-β3, the final phase of the study explored whether this scaffold could be used as a TGF-β3 delivery system to promote chondrogenesis of FPSCs. It was found that the majority of TGF-β3 that was loaded onto the scaffold was released in a controlled manner over the first 10days of culture, with comparable long-term chondrogenesis observed in these TGF-β3-loaded constructs compared to scaffolds where the TGF-β3 was continuously added to the media. The results of this study support the use of cartilage-ECM-derived scaffolds as a growth factor delivery system for use in articular cartilage regeneration.

Currently available skin substitutes are still associated with a range of problems including poor engraftment resulting from deficient vascularization, and excessive scar formation, among others. Trying to overcome these issues, this work proposes the combination of poly(3-hydroxybutyrate-co-hydroxyvalerate) (PHBV) structures with adipose-derived stem cells (ASCs) to offer biomechanical and biochemical signaling cues necessary to improve wound healing in a full-thickness model. PHBV scaffold maintained the wound moisture and demonstrated enough mechanical properties to withstand wound contraction. Also, exudate and inflammatory cell infiltration enhanced the degradation of the structure, and thus healing progression. After 28 days all the wounds were closed and the PHBV scaffold was completely degraded. The transplanted ASCs were detected in the wound area only at day 7, correlating with an up-regulation of VEGF and bFGF at this time point that consequently led to a significant higher vessel density in the group that received the PHBV loaded with ASCs. Subsequently, the dermis formed in the presence of the PHBV loaded with ASCs possesses a more complex collagen structure. Additionally, an anti-scarring effect was observed in the presence of the PHBV scaffold indicated by a down-regulation of TGF-β1 and α-SMA together with an increase of TGF-β3, when associated with ASCs. These results indicate that although PHBV scaffold was able to guide the wound healing process with reduced scarring, the presence of ASCs was crucial to enhance vascularization and provide a better quality neo-skin. Therefore, we can conclude that PHBV loaded with ASCs possesses the necessary bioactive cues to improve wound healing with reduced scarring.

Cartilage lesions in degenerative osteoarthritis (OA) are involved with pathological microenvironmental alterations induced by inflammatory macrophages, and apoptotic and/or hypertrophic chondrocytes. However, current non-operative therapies for cartilage repair in OA can rarely achieve long-term and satisfactory outcomes. This study aims to evaluate a newly developed squid type II collagen (SCII) for repairing OA-induced cartilage lesions. Our in vitro data show that SCII induces M2 polarization of macrophages, and activates macrophages to express pro-chondrogenic genes (TGF-β and IGF), which greatly improves the microenvironment around chondrocytes to produce type II collagen and glycosaminoglycan. In addition, glycine in SCII activates glycine receptors on inflammatory chondrocytes to decrease intracellular calcium concentration, leading to effective inhibition of chondrocyte apoptosis and hypertrophy. The in vitro effects of SCII are further confirmed in vivo. In a rat model of OA, SCII increases the ratio of M2 macrophages, elevates the levels of pro-chondrogenic cytokines (TGF-β1 and TGF-β3) in synovial fluid, and inhibits chondrocyte apoptosis and MMP13 production. Our findings show that SCII immunomodulates M2 activation of macrophages to skew the local OA microenvironment towards a pro-chondrogenic atmosphere, and promotes cartilage repair under inflammatory condition. It shows great potential for SCII to be a novel biomaterial for cartilage repair in OA.

TGF-β1, β2 and β3 bind a common receptor to exert vastly diverse effects in cancer, supporting either tumor progression by favoring metastases and inhibiting anti-tumor immunity, or tumor suppression by inhibiting malignant cell proliferation. Global TGF-β inhibition thus bears the risk of undesired tumor-promoting effects. We show that selective blockade of TGF-β1 production by Tregs with antibodies against GARP:TGF-β1 complexes induces regressions of mouse tumors otherwise resistant to anti-PD-1 immunotherapy. Effects of combined GARP:TGF-β1/PD-1 blockade are immune-mediated, do not require FcγR-dependent functions and increase effector functions of anti-tumor CD8⁺ T cells without augmenting immune cell infiltration or depleting Tregs within tumors. We find GARP-expressing Tregs and evidence that they produce TGF-β1 in one third of human melanoma metastases. Our results suggest that anti-GARP:TGF-β1 mAbs, by selectively blocking a single TGF-β isoform emanating from a restricted cellular source exerting tumor-promoting activity, may overcome resistance to PD-1/PD-L1 blockade in patients with cancer.

Patients with recessive dystrophic epidermolysis bullosa (RDEB) have incurable skin fragility, blistering, and skin wounds due to mutations in the gene that codes for type VII collagen (C7) that mediates dermal-epidermal adherence in human skin. In this study, we evaluated if topically applied human recombinant C7 (rC7) could restore C7 at the dermal-epidermal junction (DEJ) and enhance wound healing. We found that rC7 applied topically onto murine skin wounds stably incorporated into the newly formed DEJ of healed wounds and accelerated wound closure by increasing re-epithelialization. Topical rC7 decreased the expression of fibrogenic transforming growth factor-β2 (TGF-β2) and increased the expression of anti-fibrogenic TGF-β3. These were accompanied by the reduced expression of connective tissue growth factor, fewer α smooth muscle actin (α-SMA)-positive myofibroblasts, and less deposition of collagen in the healed neodermis, consistent with less scar formation. In addition, using a mouse model in which skin from C7 knock out mice was grafted onto immunodeficient mice, we showed that applying rC7 onto RDEB grafts with wounds restored C7 and anchoring fibrils (AFs) at the DEJ of the grafts and corrected the dermal-epidermal separation. The topical application of rC7 may be useful for treating patients with RDEB and patients who have chronic skin wounds.

Chondrogenic differentiation and cartilage tissue formation derived from stem cells are highly dependent on both biological and mechanical factors. This study investigated whether or not fibrin-hyaluronic acid (HA) coupled with low-intensity ultrasound (LIUS), a mechanical stimulation, produces an additive or synergistic effect on the chondrogenesis of rabbit mesenchymal stem cells (MSCs) derived from bone marrow. For the purpose of comparison, rabbit MSCs were first cultured in fibrin-HA or alginate hydrogels, and then subjected to chondrogenic differentiation in chondrogenic-defined medium for 4 weeks in the presence of either transforming growth factor-beta3 (TGF-β3) (10 ng/mL) or LIUS treatment (1.0 MHz and 200 mW/cm(2) ). The resulting samples were evaluated at 1 and 4 weeks by histological observation, chemical assays, and mechanical analysis. The fibrin-HA hydrogel was found to be more efficient than alginate in promoting chondrogenesis of the MSCs by producing a larger amount of sulfated glycosaminoglycans (GAGs) and collagen, and engineered constructs made with the hydrogel demonstrated higher mechanical strength. At 4 weeks of tissue culture, the chondrogenesis of the MSCs in fibrin-HA were shown to be further enhanced by treatment with LIUS, as observed by analyses for the amounts of GAGs and collagen, and mechanical strength testing. In contrast, TGF-β3, a well-known chondrogenic inducer, showed a marginal additive effect in the amount of collagen only. These results revealed that LIUS further enhanced chondrogenesis of the MSCs cultured in fibrin-HA, in vitro, and suggested that the combination of fibrin-HA and LIUS is a useful tool in constructing high-quality cartilage tissues from MSCs.

Non-coding RNAs have been less studied in cartilage development and destruction regulated by sophisticated molecular events despite their considerable theranostic potential. In this study, we identified significant down-regulation of mR-101 and up-regulation of lncRNA, HOTTIP in the processes of endochondral ossification and osteoarthritic progression. In wing mesenchymal cells, up-expression of miR-101 by TGF-β3 treatment is targeting DNMT-3B and thereby altered the methylation of integrin-α1 addressed as a positive regulator of endochondral ossification in this study. In like manner, down-regulation of miR-101 also coordinately up-regulated DNMT-3B, down-regulated integrin-α1, and resulted in cartilage destruction. In an OA animal model, introduction of lentiviruses that encoded miR-101 or integrin-α1 successfully reduced cartilage destruction. In like manner, long non-coding RNA (lncRNA), HOTTIP, a known regulator for HoxA genes, was highly up-regulated and concurrent down-regulation of HoxA13 displayed the suppression of integrin-α1 in OA chondrocytes. In conclusion, two non-coding RNAs, miR-101 and HOTTIP regulate cartilage development and destruction by modulating integrin-α1 either epigenetically by DNMT-3B or transcriptionally by HoxA13 and data further suggest that these non-coding RNAs could be a potent predictive biomarker for OA as well as a therapeutic target for preventing cartilage-related diseases.

Adipose-derived stem cells (ASCs) have captured growing interests for cartilage regeneration. Although ASCs chondrogenesis can be stimulated by genetic modification, whether genetically engineered ASCs hold promise for the cartilaginous tissue formation remains to be explored. Since baculovirus (an emerging gene delivery vector) effectively transduced ASCs and transforming growth factor β3 (TGF-β3) was recently shown to induce ASCs chondrogenesis more potently than TGF-β1, we constructed a baculoviral vector (Bac-CT3W) to encode TGF-β3. The Bac-CT3W-transduced ASCs expressed TGF-β3 robustly and substantiated the chondrogenesis of ASCs cultured in monolayer and in porous scaffolds. Culture of the transduced cell/scaffold constructs in the rotating-shaft bioreactor (RSB) under hypoxic and perfusion conditions for 2 weeks further augmented the ASCs chondrogenesis and deposition of cartilage-specific collagen II and glycosaminoglycans, leading to the formation of cartilage-like tissues with hyaline appearance and compressive modulus approaching 62% of the native articular cartilage. Intriguingly, prolonged culture to 3 or 4 weeks failed to further augment the construct growth, probably due to the scaffold degradation. Altogether, baculovirus-mediated TGF-β3 expression in ASCs in conjunction with dynamic culture in the RSB for 2 weeks synergistically ameliorated the ASCs chondrogenesis and formation of cartilaginous tissues, representing a novel approach to producing engineered cartilages.

Collagen XXIV (Col24α1) is a recently discovered fibrillar collagen. It is known that mouse Col24α1 is predominantly expressed in the forming skeleton of the mouse embryo, as well as in the trabecular bone and periosteum of the newborn mouse. However, the role and mechanism of Col24α1 in osteoblast differentiation and mineralization remains unclear. By analyzing the expression pattern of Col24α1, we confirmed that it is primarily expressed in bone tissues, and this expression gradually increased concomitant with the progression of osteoblast differentiation. Through the use of a lentivirus vector-mediated interference system, silencing Col24α1 expression in MC3T3-E1 murine preosteoblastic cells resulted in significant inhibition of alkaline phosphatase (ALP) activity, cell mineralization, and the expression of osteoblast marker genes such as runt-related transcription factor 2 (Runx2), osteocalcin (OCN), ALP, and type I collagen (Col I). Subsequent overexpression not only rescued the deficiency in osteoblast differentiation from Col24α1 silenced cells, but also enhanced osteoblastic differentiation in control cells. We further revealed that Col24α1 interacts with integrin β3, and silencing Col24α1 up-regulated the expression of Smad7 during osteoblast differentiation while at the same time inhibiting the phosphorylation of the Smad2/3 complex. These results suggest that Col24α1 imparts some of its regulatory control on osteoblast differentiation and mineralization at least partially through interaction with integrin β3 and the transforming growth factor beta (TGF-β) /Smads signaling pathway.

BACKGROUND

Numerous experimental efforts have been undertaken to induce the healing of lesions within articular cartilage by re-establishing competent repair tissue. Adult mesenchymal stem cells have attracted attention as a source of cells for cartilage tissue engineering. The purpose of this study was to investigate chondrogenesis employing periosteal mesenchymal cells.

METHODS

Periosteum was harvested from patients who underwent orthopedic surgeries. Mesenchymal stem cells were characterized through flow cytometry using specific antibodies. The stem cells were divided into four groups. Two groups were stimulated with transforming growth factor β3 (TGF-β3), of which one group was cultivated in a monolayer culture and the other was cultured in a micromass culture. The remaining two groups were cultivated in monolayer or micromass cultures in the absence of TGF-β3. Cell differentiation was verified through quantitative reverse transcription-polymerase chain reaction (RT-PCR) and using western blot analysis.

RESULTS

In the groups cultured without TGF-β3, only the cells maintained in the micromass culture expressed type II collagen. Both the monolayer and the micromass groups that were stimulated with TGF-β3 expressed type II collagen, which was observed in both quantitative RT-PCR and western blot analysis. The expression of type II collagen was significantly greater in the micromass system than in the monolayer system.

CONCLUSIONS

The results of this study demonstrate that the interactions between the cells in the micromass culture system can regulate the proliferation and differentiation of periosteal mesenchymal cells during chondrogenesis and that this effect is enhanced by TGF-β3.

Spermatogenesis takes place in the seminiferous tubules in adult testes such as rats, in which developing germ cells must traverse the seminiferous epithelium while spermatogonia (2n, diploid) undergo mitotic and meiotic divisions, and differentiate into elongated spermatids (1n, haploid). It is conceivable that this event involves extensive junction restructuring particularly at the blood-testis barrier (BTB, a structure that segregates the seminiferous epithelium into the basal and the adluminal compartments) that occurs at stages VII-VIII of the seminiferous epithelial cycle. As such, cross-talk between tight (TJ) and anchoring junctions [e.g., basal ectoplasmic specialization (basal ES), adherens junction (AJ), desmosome-like junction (DJ)] at the BTB must occur to coordinate the transient opening of the BTB to facilitate preleptotene spermatocyte migration. Interestingly, while there are extensively restructuring at the BTB during the epithelial cycle, the immunological barrier function of the BTB must be maintained without disruption even transiently. Recent studies using the androgen suppression and Adjudin models have shown that anchoring junction restructuring that leads to germ cell loss from the seminiferous epithelium also promotes the production of AJ (e.g., basal ES) proteins (such as N-cadherins, catenins) at the BTB site. We postulate the testis is using a similar mechanism during spermatogenesis at stage VIII of the epithelial cycle that these induced basal ES proteins, likely form a "patch" surrounding the BTB, transiently maintain the BTB integrity while TJ is "opened", such as induced by TGF-b3 or TNFa, to facilitate preleptotene spermatocyte migration. However, in other stages of the epithelial cycle other than VII and VIII when the BTB remains "closed" (for approximately 10 days), anchoring junctions (e.g., AJ, DJ, and apical ES) restructuring continues to facilitate germ cell movement. Interestingly, the mechanism(s) that governs this communication between TJ and anchoring junction (e.g., basal ES and AJ) in the testis has remained obscure until recently. Herein, we provide a critical review based on the recently available data regarding the cross-talk between TJ and anchoring junction to allow simultaneous maintenance of the BTB and germ cell movement across the seminiferous epithelium.

BACKGROUND

Proinflammatory cytokines induce the expression of matrix metalloproteinases that play a crucial role in myocardial remodeling. Beta-adrenergic receptor stimulation influences the production of cytokines heralding the possibility of modulating cytokine production by beta-adrenergic blockers.

RESULTS

In a coxsackievirus B3 murine myocarditis model (BALB/c), effects of carvedilol and metoprolol on myocardial cytokine expression, inflammatory cell infiltration and MMP/TIMP profiles were investigated. In carvedilol-treated mice, a significant improvement in left ventricular function was documented 10 days post infection. In infected mice (n=10), IL-1beta, TNF-alpha, TGF-beta(1) and IL-10 myocardial mRNA abundance were increased significantly (240%, 200%, 161%, and 230%) compared to controls (n=10), while IL-15 mRNA was markedly reduced (70%). Infected mice showed significantly increased infiltrations with CD3-, CD4- and CD8-T-lymphocytes (730%, 1110%, 380%). In the infected mice, myocardial MMP/TIMP profiles presented a significant upregulation of membrane type-1 MMP, MMP-9, MMP-8 and MMP-3 (150%, 160%, 340%, and 270%) and a significant decrease in TIMP-4 levels (75%). Carvedilol attenuated over-expression of myocardial TGF-beta(1), IL-1beta and MMP-8 mRNA expression significantly and induced a relevant IL-10 mRNA expression in the infected mice (n=10). By an unchanged infiltration with CD3-T-lymphocytes, carvedilol showed a representative reduction in CD4-T-lymphocytes.

CONCLUSIONS

Carvedilol treatment in experimental myocarditis leads to reduced expression of proinflammatory cytokines and MMPs, which contributes to reduced matrix degradation and ultimately to improved structural integrity of the heart. Besides the antiadrenergic potential, carvedilol is beneficial due to a wide range of biological activities (antiinflammatory, antifibrotic, antioxidative and immunomodulatory).

Recapitulation of human corneal stromal tissue is believed to be among the most challenging steps in engineering human corneal tissue because of the difficulty in reproducing its highly-ordered hierarchical ultrastructure, which imparts its robust biomechanical properties and optical transparency. In this study, we compared the feasibility of utilizing human corneal stromal stem cells (hCSSCs) and human corneal fibroblasts (hCFs) in the generation of human corneal stromal tissue on a highly-aligned fibrous substrate made from poly(ester urethane) urea. In the serum-free keratocyte differentiation medium supplemented with FGF-2 (10 ng/mL) and TGF-β3 (0.1 ng/mL), hCSSCs successfully differentiated into keratocytes and secreted multilayered lamellae with orthogonally-oriented collagen fibrils, in a pattern mimicking human corneal stromal tissue. The constructs were 60-70 μm thick and abundant in cornea-specific extracellular matrix (ECM) components, including keratan sulfate, lumican, and keratocan. Under the identical conditions, hCFs tended to differentiate into myofibroblasts and deposited a less-organized collagen-fibrillar construct in a pattern with similarities to corneal scar tissue due to a lack of cornea-specific ECM components. These observations demonstrated that hCSSCs showed a much greater potential, under proper substrate and growth factor guidance, to facilitate the generation of a biological human cornea equivalent. Unlike hCSSCs, hCFs were less responsive to these environmental cues and under identical culture conditions generated an ECM that poorly mimicked the native, functional tissue structure and composition.

T helper 17 (Th17) cells play critical roles in inflammatory and autoimmune diseases. The lineage-specific transcription factor RORγt is the key regulator for Th17 cell fate commitment. A substantial number of studies have established the importance of transforming growth factor β (TGF-β) -dependent pathways in inducing RORγt expression and Th17 differentiation. TGF-β superfamily members TGF-β1 , TGF-β3 or activin A, in concert with interleukin-6 or interleukin-21, differentiate naive T cells into Th17 cells. Alternatively, Th17 differentiation can occur through TGF-β-independent pathways. However, the mechanism of how TGF-β-dependent and TGF-β-independent pathways control Th17 differentiation remains controversial. This review focuses on the perplexing role of TGF-β in Th17 differentiation, depicts the requirement of TGF-β for Th17 development, and underscores the multiple mechanisms underlying TGF-β-promoted Th17 generation, pathogenicity and plasticity. With new insights and comprehension from recent findings, this review specifically tackles the involvement of the canonical TGF-β signalling components, SMAD2, SMAD3 and SMAD4, summarizes diverse SMAD-independent mechanisms, and highlights the importance of TGF-β signalling in balancing the reciprocal conversion of Th17 and regulatory T cells. Finally, this review includes discussions and perspectives and raises important mechanistic questions about the role of TGF-β in Th17 generation and function.

This study aimed to investigate the synergy between transforming growth factor beta 3 (TGF-β3) and insulin-like growth factor 1 (IGF-1) on nucleus pulposus-derived mesenchymal stem cells (NP-MSCs) and the underlying mechanism using a serum-free culture system. NP-MSC proliferation and viability were measured using a CCK-8 assay and annexin V-FITC/propidium iodide, respectively. NP-MSCs in micromasses were investigated for differentiation towards nucleus pulposus cells (NPCs). SOX-9, collagen-I, collagen-II, aggrecan and decorin expressions were detected by RT-PCR and immunoblotting. Matrix deposition was assessed by sulfated glycosaminoglycan (sGAG) analysis. Novel chondrogenic and nucleus pulposus (NP) genes were detected to distinguish differentiated cell types. MAPK/ERK and TGF/Smad signaling pathways were also examined. As a result, the synergy between TGF-β3 and IGF-1 enhanced NP-MSC viability, extracellular matrix (ECM) biosynthesis and differentiation towards NPCs, partly through the activation of the MAPK/ERK signaling pathway. Therefore, the synergy between TGF-β3 and IGF-1 ameliorates NP-MSC viability, differentiation and promotes intervertebral disc regeneration.

There is an unmet need for improved, effective tissue engineering strategies to replace or repair bone damaged through disease or injury. Recent research has focused on developing biomaterial scaffolds capable of spatially and temporally releasing combinations of bioactive growth factors, rather than individual molecules, to recapitulate repair pathways present in vivo. We have developed an ex vivo embryonic chick femur critical size defect model and applied the model in the study of novel extracellular matrix (ECM) hydrogel scaffolds containing spatio-temporal combinatorial growth factor-releasing microparticles and skeletal stem cells for bone regeneration. Alginate/bovine bone ECM (bECM) hydrogels combined with poly(d,l-lactic-co-glycolic acid) (PDLLGA)/triblock copolymer (10-30% PDLLGA-PEG-PLDLGA) microparticles releasing dual combinations of vascular endothelial growth factor (VEGF), chondrogenic transforming growth factor beta 3 (TGF-β3) and the bone morphogenetic protein BMP2, with human adult Stro-1+bone marrow stromal cells (HBMSCs), were placed into 2mm central segmental defects in embryonic day 11 chick femurs and organotypically cultured. Hydrogels loaded with VEGF combinations induced host cell migration and type I collagen deposition. Combinations of TGF-β3/BMP2, particularly with Stro-1+HBMSCs, induced significant formation of structured bone matrix, evidenced by increased Sirius red-stained matrix together with collagen expression demonstrating birefringent alignment within hydrogels. This study demonstrates the successful use of the chick femur organotypic culture system as a high-throughput test model for scaffold/cell/growth factor therapies in regenerative medicine. Temporal release of dual growth factors, combined with enriched Stro-1+HBMSCs, improved the formation of a highly structured bone matrix compared to single release modalities. These studies highlight the potential of a unique alginate/bECM hydrogel dual growth factor release platform for bone repair.