OBJECTIVE

We showed previously that single nucleotide polymorphism (SNP) rs662702 in PAX6 may be located in a microRNA-328 binding site that causes susceptibility to high myopia. Our study was done to elucidate the role of PAX6 and its relationship with microRNA-328 in myopia.

METHODS

A luciferase assay was used to confirm microRNA-328 binding to the PAX6 locus. Clones containing each allele of rs662702 were created and tested for their binding affinity to microRNA-328. Because a low level of PAX6 is a risk factor for myopia, we tested whether knockdown of PAX6 affects retinal pigment epithelial (RPE) cells and scleral cells, as well as expression of myopia-related genes. We also tested for the effect of retinoic acid (RA) on microRNA-328 expression, since RA-responsive elements are predicted to lie in the microRNA-328 promoter.

RESULTS

MicroRNA-328 was shown to bind to the wild-type, but not mutant 3' untranslated region (UTR) of PAX6. The risk C allele of rs644242 had strong response to microRNA-328 but the protective T allele did not respond to microRNA-328. Down-regulation of PAX6 in RPE increased RPE proliferation, but reduced scleral cell proliferation. In addition, transforming growth factor (TGF)-β3 in the RPE and matrix malleoproteinase-2 (MMP2) in the sclera were increased, while collagen I and integrin β1 in the sclera were decreased. RA dose-dependently increased microRNA-328 expression and, in turn, suppressed PAX6 expression.

CONCLUSIONS

We elaborated the relationship among myopia development, SNP rs662702, microRNA-328 and RA. The data imply that reduction of miR-328 and/or RA can be potential strategies for myopia prevention or treatment.

Transforming growth factor β (TGF-β) promotes tissue fibrosis via the receptor-specific Smad pathway and non-canonical pathways. We recently reported that TGF-β1-stimulated collagen expression by cultured kidney cells requires integrin-dependent activation of focal adhesion kinase (FAK) and consequent ERK MAP kinase activity leading to Smad3 linker region phosphorylation. Here, we defined a role for αvβ3-integrin in this non-canonical pathway. A human kidney tubular cell line in which β1-integrin was knocked down (β1-k/d) demonstrated enhanced type I collagen mRNA expression and promoter activity. A second shRNA to either αv-integrin or β3-integrin, but not to another αv-binding partner, β6-integrin, abrogated the enhanced COL1A2 promoter activity in β1-k/d cells. Although αvβ3-integrin surface expression levels were not different, αvβ3-integrins colocalized with sites of focal adhesion significantly more in β1-k/d cells, and activated αvβ3-integrin was detected only in β1-k/d cells. Further, the collagen response was decreased by a function-blocking antibody or a peptide inhibitor of αvβ3-integrin. In cells lacking αvβ3-integrin, the responses were attenuated, whereas the response was enhanced in αvβ3-overexpressing cells. Rac1 and ERK, previously defined mediators for this non-canonical pathway, showed increased activities in β1-k/d cells. Finally, inhibition of αvβ3-integrin decreased Rac1 activity and COL1A2 promoter activity in β1-k/d cells. Together, our results indicate that decreasing β1 chain causes αvβ3-integrin to become functionally dominant and promotes renal cell fibrogenesis via Rac1-mediated ERK activity.

Exposure to excess testosterone (T) during fetal life has a profound impact on the metabolic and reproductive functions in the female's postnatal life. However, less is known about the effects of excess testosterone in males. The aim of the present study was to evaluate the impact (consequences) of an excess of T during fetal development on mature male testis. The testicular evaluation was by histological analysis and by determination of mRNA expression of the FSH receptor (FSH-R), transforming growth factor-β type I receptor (TβR-I), and two members of the TGF-β superfamily, transforming growth factor-β3 (TGFβ3) and anti-Müllerian hormone (AMH) in males born to mothers receiving an excess of T during pregnancy. At 42 wk of age, postpubertal males born to mothers treated with 30 mg of T propionate twice weekly from day 30 to 90, followed by 40 mg of T propionate from day 90 to 120 of pregnancy (T males), showed higher concentrations of FSH in response to a GnRH analog, a higher number of Sertoli cells/seminiferous tubule cross-section, and a lower number of germ cells/tubules (P < 0.05) than control males (C males) born to mothers treated with the vehicle. The mRNA expression of FSH-R and of TβR-I was higher in T males compared with C males (P < 0.05). Moreover, in T males, AMH expression level correlated negatively with the expression level of TGFβ3. In C males, this latter correlation was not observed. These results suggest that prenatal exposure to an excess of T can negatively modify some histological and molecular characteristics of the mature testis.

BACKGROUND

Keloids are the result of excessive scar tissue formation. Besides their poor aesthetic appearance, keloids can be associated with severe clinical symptoms such as pain, itching, and rigidity. Unfortunately, most therapeutic approaches remain clinically unsatisfactory. Recently, injections with botulinum toxin A (BTA) were proposed for the treatment of established keloids in a clinical trial. In this study, we aimed to verify the effects of intralesional BTA for the treatment of therapy-resistant keloids using objective measurements. In addition, the underlying molecular mechanisms were investigated using cultured keloid-derived fibroblasts.

METHODS

Four patients received BTA (doses varying from 70 to 140 Speywood units per session) injected directly into their keloids every 2 months for up to 6 months. Differences in height and volume were evaluated clinically and measured with a 3-D optical profiling system. Keloid-derived fibroblasts were treated with different concentrations of BTA, and expression of collagen (COL)1A1, COL1A2, COL3A1, TGF-β1, TGF-β2, TGF-β3, fibronectin-1, laminin-β2, and α-SMA was determined by real-time quantitative PCR. MTT and BrdU assays were used to analyze the effects of BTA on fibroblast proliferation and metabolism.

RESULTS

Intralesional administration of BTA did not result in regression of keloid tissue. No differences in expression of ECM markers, collagen synthesis, or TGF-β could be observed after BTA treatment of keloid fibroblasts. In addition, cell proliferation and metabolism of keloid fibroblasts was not affected by BTA treatment.

CONCLUSIONS

The suggested clinical efficiency of intralesional BTA for the therapy of existent keloids could not be confirmed in this study. Based on our data, the potential mechanisms of action of BTA on keloid-derived fibroblasts remain unclear.

BACKGROUND

Colorectal cancer (CRC) is characterised by hypoxia, which activates gene transcription through hypoxia-inducible factors (HIF), as well as by expression of epidermal growth factor (EGF) and EGF receptors, targeting of which has been demonstrated to provide therapeutic benefit in CRC. Although EGF has been demonstrated to induce expression of angiogenic mediators, potential interactions in CRC between EGF-mediated signalling and the hypoxia/HIF pathway remain uncharacterised.

METHODS

PCR-based profiling was applied to identify angiogenic genes in Caco-2 CRC cells regulated by hypoxia, the hypoxia mimetic dimethyloxallylglycine (DMOG) and/or EGF. Western blotting was used to determine the role of HIF-1alpha, HIF-2alpha and MAPK cell signalling in mediating the angiogenic responses.

RESULTS

We identified a total of 9 angiogenic genes, including angiopoietin-like (ANGPTL) 4, ephrin (EFNA) 3, transforming growth factor (TGF) β1 and vascular endothelial growth factor (VEGF), to be upregulated in a HIF dependent manner in Caco-2 CRC cells in response to both hypoxia and the hypoxia mimetic dimethyloxallylglycine (DMOG). Stimulation with EGF resulted in EGFR tyrosine autophosphorylation, activation of p42/p44 MAP kinases and stabilisation of HIF-1α and HIF-2α proteins. However, expression of 84 angiogenic genes remained unchanged in response to EGF alone. Crucially, addition of DMOG in combination with EGF significantly increased expression of a further 11 genes (in addition to the 9 genes upregulated in response to either DMOG alone or hypoxia alone). These additional genes included chemokines (CCL-11/eotaxin-1 and interleukin-8), collagen type IV α3 chain, integrin β3 chain, TGFα and VEGF receptor KDR.

CONCLUSIONS

These findings suggest that although EGFR phosphorylation activates the MAP kinase signalling and promotes HIF stabilisation in CRC, this alone is not sufficient to induce angiogenic gene expression. In contrast, HIF activation downstream of hypoxia/DMOG drives expression of genes such as ANGPTL4, EFNA3, TGFβ1 and VEGF. Finally, HIF activation synergises with EGF-mediated signalling to additionally induce a unique sub-group of candidate angiogenic genes. Our data highlight the complex interrelationship between tumour hypoxia, EGF and angiogenesis in the pathogenesis of CRC.

The signaling pathway of transforming growth factor β2 (TGF-β2)/Smad plays an important role in the pathological process in posterior capsule opacification (PCO) after cataract surgery. Smad2 and Smad3 are both receptor-regulated Smads (R-Smads) of the TGF-β2 signaling pathway. We aim to find which among Smad2, Smad3, and Smad2&3 plays a key role in PCO pathology. The signal characteristics of TGF-β2 and Smad proteins in the human lens cell line HLE-B3 were investigated. Smad2, Smad3, or Smad2&3 were silenced using small interfering RNA. We then tested cell proliferation by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and cell growth curve assays, migration by transwell and wound-healing assays, and extracellular matrix production including α-smooth muscle actin (αSMA), fibronectin, and type I collagen by real-time PCR assay, with and without TGF-β2 exposure. Silencing Smad3 blocked the effect of TGF-β2 on cell proliferation and production of fibronectin and type I collagen. Silencing Smad2 blocked the effect of TGF-β2 on cell migration and production of αSMA. Smad2 depletion enhanced Smad3 activity in cell proliferation and ECM production, whereas Smad3 depletion enhanced Smad2 activity in migration and αSMA expression. Silencing Smad2 and Smad3 efficiently blocked the effect of TGF-β2on cell proliferation, migration, and extracellular matrix production. Smad2 and Smad3 are both key in the TGF-β2 signaling pathway. We can prevent the development of PCO following cataract surgery by blocking the TGF-β2/Smad2&3 signaling pathway.

Aldose reductase (AR) and TGF-beta have been implicated in the development of diabetic complications, such as cataracts. In an attempt to obtain potential agents for the prevention of diabetic cataracts from natural products, we purified genistein from the roots of Pueraria lobata and investigated its inhibitory effects upon AR activity and its antioxidant effects on rat lenses. The inhibition of AR activity by genistein increased in a dose-dependent manner and the opacities of lenses were significantly improved when treated with genistein. In addition, we determined the effects of genistein on mechanisms induced by exposure to high glucose in human lens epithelial (HLE-B3) cells. We found that genistein was able to reduce the expression of TGF-beta2, alphaB-crystallin, and fibronectin mRNAs in HLE-B3 cells that were cultured in high glucose conditions. In addition, a reduction in glutathione (GSH) levels and thiobarbituratic acid-reactive substances was observed. These results show that genistein is protective against lens opacity and also inhibits high glucose-mediated toxic effects in HLE-B3 cells. These effects are likely achieved by preventing AR and cellular oxidation; therefore, genistein may be a potential therapeutic agent for preventing and treating complications associated with diabetes mellitus, such as diabetic cataracts.

In this work, it was hypothesized that co-cultures of articular chondrocytes (ACs) and mesenchymal stem cells (MSCs) would exhibit enhanced sensitivity to chondrogenic stimuli, such as TGF-β3, and would require a reduced concentration of TGF-β3 to achieve an equivalent level of chondrogenesis compared to monocultures of each cell type. Furthermore, it was hypothesized that compared to monocultures, the chondrogenic phenotype of AC/MSC co-cultures would be more stable upon the removal of TGF-β3 from the culture medium. These hypotheses were investigated by culturing ACs and MSCs alone and in a 1:3 ratio on electrospun poly(ε-caprolactone) scaffolds. All cell populations were cultured for two weeks with 0, 1, 3, or 10 ng/ml of TGF-β3. After two weeks growth factor supplementation was removed, and the constructs were cultured for two additional weeks. Cell proliferation, extracellular matrix production, and chondrogenic gene expression were evaluated after two and four weeks. The results demonstrated that co-cultures of ACs and MSCs require a reduced concentration and duration of TGF-β3 exposure to achieve an equivalent level of chondrogenesis compared to AC or MSC monocultures. Thus, the present work implicates that the promise of co-cultures for cartilage engineering is enhanced by their robust phenotype and heightened sensitivity to TGF-β3.

Our previous study showed that miR-29 attenuates muscle wasting in chronic kidney disease. Other studies found that miR-29 has anti-fibrosis activity. We hypothesized that intramuscular injection of exosome-encapsulated miR-29 would counteract unilateral ureteral obstruction (UUO)-induced muscle wasting and renal fibrosis. We used an engineered exosome vector, which contains an exosomal membrane protein gene Lamp2b that was fused with the targeting peptide RVG (rabies viral glycoprotein peptide). RVG directs exosomes to organs that express the acetylcholine receptor, such as kidney. The intervention of Exo/miR29 increased muscle cross-sectional area and decreased UUO-induced upregulation of TRIM63/MuRF1 and FBXO32/atrogin-1. Interestingly, renal fibrosis was partially depressed in the UUO mice with intramuscular injection of Exo/miR29. This was confirmed by decreased TGF-β, alpha-smooth muscle actin, fibronectin, and collagen 1A1 in the kidney of UUO mice. When we used fluorescently labeled Exo/miR29 to trace the Exo/miR route in vivo and found that fluorescence was visible in un-injected muscle and in kidneys. We found that miR-29 directly inhibits YY1 and TGF-β3, which provided a possible mechanism for inhibition of muscle atrophy and renal fibrosis by Exo/miR29. We conclude that Exo/miR29 ameliorates skeletal muscle atrophy and attenuates kidney fibrosis by downregulating YY1 and TGF-β pathway proteins.

It has been recently ascribed to several inflammatory cytokines (i.e. TGF-β3, TNF-α, and IL-1) a functional role in regulating Sertoli cell blood-testis barrier (BTB) dynamics. In the testis, IL-6 inhibits meiotic DNA synthesis during the seminiferous epithelium cycle, reduces sperm motility and influences the secretion of transferrin and inhibin B by Sertoli cells. Also, it has been shown that IL-6 affects tight junction permeability in Sertoli cells, but, little is known about its role in regulating the BTB. The aim of this study was to investigate the molecular mechanisms by which IL-6 affects BTB dynamics. We show that IL-6 perturbs the integrity of the BTB, and alters the normal localization and steady-state levels of BTB integral membrane proteins. We demonstrated that IL-6 regulates the BTB by inhibiting the degradation of BTB constitutive proteins and activating ERK-MAPK pathways. Our results provide mechanistic insight into the roles of IL-6 in regulating BTB dynamics.

Chronic liver injury can result in fibrosis that may progress over years to end-stage liver disease. The most effective anti-fibrotic therapy is treatment of the underlying disease, however when not possible, interventions to reverse or slow fibrosis progression are needed.

The aim of this study was to study the safety and tolerability of simtuzumab, a monoclonal antibody directed against lysyl oxidase-like 2 (LOXL2) enzyme, in subjects with hepatitis C virus (HCV), human immunodeficiency virus (HIV), or HCV-HIV co-infection and advanced liver disease.

Eighteen subjects with advanced liver fibrosis received simtuzumab 700 mg intravenously every 2 weeks for 22 weeks. Transjugular liver biopsies were performed during screening and at the end of treatment to measure hepatic venous pressure gradient (HVPG) and to stage fibrosis.

Treatment was well-tolerated with no discontinuations due to adverse events. No significant changes were seen in HVPG or liver biopsy fibrosis score after treatment. Exploratory transcriptional and protein profiling using paired pre- and post-treatment liver biopsy and serum samples suggested up-regulation of TGF-β3 and IL-10 pathways with treatment.

In this open-label, pilot clinical trial, simtuzumab treatment was well-tolerated in HCV- and HIV-infected subjects with advanced liver disease. Putative modulation of TGF-β3 and IL-10 pathways during simtuzumab treatment merits investigation in future trials.

OBJECTIVE

Repairing articular cartilage is clinically challenging. We investigated a simple, effective and clinically feasible cell-based therapeutic approach using a poly(lactide-co-glycolide) (PLGA) scaffold seeded with autologous endothelial progenitor cells (EPC) to repair a full-thickness osteochondral defect in rabbits using a one-step surgery.

METHODS

EPC obtained by purifying a small amount of peripheral blood from rabbits were seeded into a highly porous, biocompatible PLGA scaffold, namely, EPC-PLGA, and implanted into the osteochondral defect in the medial femoral condyle. Twenty two rabbits were randomized into one of three groups: the empty defect group (ED), the PLGA-only group or the EPC-PLGA group. The defect sites were evaluated 4 and 12 weeks after implantation.

RESULTS

At the end of testing, only the EPC-PLGA group showed the development of new cartilage tissue with a smooth, transparent and integrated articular surface. Moreover, histological analysis showed obvious differences in cartilage regeneration. At week 4, the EPC-PLGA group showed considerably higher TGF-β2 and TGF-β3 expression, a greater amount of synthesized glycosaminoglycan (GAG) content, and a higher degree of osteochondral angiogenesis in repaired tissues. At week 12, the EPC-PLGA group showed enhanced hyaline cartilage regeneration with a normal columnar chondrocyte arrangement, higher SOX9 expression, and greater GAG and collagen type II (COLII) content. Moreover, the EPC-PLGA group showed organized osteochondral integration, the formation of vessel-rich tubercular bone and significantly higher bone volume per tissue volume and trabecular thickness (Tb.Th).

CONCLUSIONS

The present EPC-PLGA cell delivery system generates a suitable in situ microenvironment for osteochondral regeneration without the supplement of exogenous growth factors.

BACKGROUND

Intravenous infusion of human amniotic epithelial cells (hAECs) has been shown to ameliorate hepatic fibrosis in murine models. Hepatic stellate cells (HSCs) are the principal collagen-secreting cells in the liver. The aim of this study was to investigate whether factors secreted by hAECs and present in hAEC-conditioned medium (CM) have anti-fibrotic effects on activated human HSCs.

METHODS

Human AECs were isolated from the placenta and cultured. Human hepatic stellate cells were exposed to hAEC CM to determine potential anti-fibrotic effects.

RESULTS

HSCs treated for 48 h with hAEC CM displayed a significant reduction in the expression of the myofibroblast markers α-smooth muscle actin and platelet-derived growth factor. Expression of the pro-fibrotic cytokine transforming growth factor-β1 (TGF-β1) and intracellular collagen were reduced by 45% and 46%, respectively. Human AEC CM induced HSC apoptosis in 11.8% of treated cells and reduced HSC proliferation. Soluble human leukocyte antigen-G1, a hAEC-derived factor, significantly decreased TGF-β1 and collagen production in activated HSCs, although the effect on collagen production was less than that of hAEC CM. The reduction in collagen and TGF-B1 could not be attributed to PGE2, relaxin, IL-10, TGF-B3, FasL or TRAIL.

CONCLUSIONS

Human AEC CM treatment suppresses markers of activation, proliferation and fibrosis in human HSCs as well as inducing apoptosis and reducing proliferation. Human AEC CM treatment may be effective in ameliorating liver fibrosis and warrants further study.

BACKGROUND

Aqueous flare as determined by laser flare photometry in the anterior chamber is a strong preoperative predictor for proliferative vitreoretinopathy (PVR) in patients with primary retinal detachment (RD). We analysed various cytokines in aqueous humour samples in relation to aqueous flare and postoperative PVR incidence in patients with RD.

METHODS

Preoperatively, the aqueous flare of patients with RD was measured quantitatively with a laser flare metre and aqueous humour samples were collected and analysed for interferon γ, tumour necrosis factor α, monocyte chemoattractant protein (MCP)-1, interleukin (IL)-1β, IL-2, IL-4, IL-6, IL-8, vascular endothelial growth factor (VEGF)-A, platelet derived growth factor (PDGF)-aa, transforming growth factor (TGF)-β1, TGF-β2, TGF-β3, fibroblast growth factor (FGF)-aa and FGF-bb by multiplex fluorescent bead-based immunoassays. Three months after RD surgery patients were examined for PVR development.

RESULTS

Of 67 consecutive patients, 10 developed at least PVR grade C. Patients with flare values >15 pc/ms (n=20) and the 10 patients with postoperative PVR all had significantly elevated levels of IL-6, IL-8, MCP-1 and TGF-β1 in aqueous humour (p≤0.05). Levels of VEGF-A, PDGF-aa and TGF-β2 were not significantly changed. Other cytokines were below the detection threshold. Eight of the 10 patients (80%) with PVR had elevated flare values of >15 pc/ms and 8 of the 20 patients (40%) with flare >15 pc/ms developed PVR. The OR for PVR with flare values >15 pc/ms was 30.7 (p=0.0001).

CONCLUSIONS

Laser flare photometry allows simple risk estimation for later PVR development. Elevated laser flare values correspond to an altered profibrotic intraocular cytokine milieu. These factors therefore constitute promising targets for a prophylactic intervention.

OBJECTIVE

Abnormality of immune regulation exists in multiple myeloma (MM). Mesenchymal stem cells (MSCs), a key regulator for immunomodulatory function, have decreased osteogenic potential in MM patients. Here we investigated the immunomodulatory function of MSCs from MM patients (MM-MSCs) and its relationship with decreased osteogenic potential.

METHODS

Real-time PCR was performed to detect the cytokines expressed in MM-MSCs (n = 22) and MSCs from normal donors (ND-MSCs, n = 11). Lymphocyte proliferative assay was used to detect the effect of MSCs on T cell proliferation. The effect of MSCs on T-cell cycle and T-cell activation markers expression were analyzed by flow cytometry. Flow cytometry and Western blot were used to detect apoptosis of T cells. Influence of T cells on osteogenic potential of MSCs was detected.

RESULTS

MM-MSCs exhibited increased expression of TGF-β1, IL-6, IL-3, TNF-α and RANKL and decreased expression of TGF-β2, TGF-β3 and FasL. The inhibitory effect of MM-MSCs on T.cell proliferative ability was attenuated. ND-MSCs silence more T cells in G0/G1 phase than MM-MSCs. The apoptosis-promoting effect of MM-MSCs on T cells seemed to be dampened. Expression of T-cell activation markers was significantly inhibited by ND-MSCs. T cells from normal donors possessed the ability to promote osteoblastic differentiation of ND-MSCs, but this ability of T cells both directly from MM patients and co-cultured with MM-MSCs was impaired.

CONCLUSIONS

MSCs from MM patients showed impaired immunoinhibitory capability on T cells, which in turn lose the ability to stimulate osteogenesis of MSCs.

BACKGROUND

Skin scarring is associated with psychosocial distress and has a negative effect on quality of life. The transforming growth factor (TGF)-β family of cytokines plays a key role in scarring. TGF-β3 improves scar appearance in a range of mammalian species. This study was performed to assess the efficacy of intradermal avotermin (TGF-β3) for the improvement of scar appearance following scar revision surgery.

METHODS

Sixty patients (35 men and 25 women; age, 19 to 78 years; 53 Caucasians; scar length, 5 to 21 cm) received intradermal avotermin (200 ng/100 μl/linear cm wound margin) and placebo to outer wound segments immediately after, and again 24 hours after, complete (group 1) or staged (group 2) scar revision surgery. A within-patient design was chosen to control for interindividual factors that affect scarring. The primary efficacy variable was a total scar score derived from a visual analogue scale, scored by a lay panel from standardized photographs from months 1 through 7 following treatment.

RESULTS

: Primary endpoint data from the combined surgical groups showed that avotermin significantly improved scar appearance compared with placebo (total scar score difference, 21.93 mm; p = 0.04). Profilometry showed a greater reduction in scar surface area from baseline with avotermin treatment compared with placebo, significant in group 2 at months 7 and 12 (difference, 41.99 mm and 25.85 mm, respectively; p = 0.03 for both comparisons). Histologic analysis from group 2 showed that, compared with placebo treatment, collagen organization in avotermin-treated scars more closely resembled normal skin in 14 of 19 cases. Avotermin was well tolerated.

CONCLUSIONS

Avotermin administration following scar revision surgery is well tolerated and significantly improves scar appearance compared with placebo.

METHODS

Therapeutic, I.(Figure is included in full-text article.).

FOXE1 mutations cause the Bamforth-Lazarus syndrome characterized by thyroid and craniofacial defects. Although a pioneer activity of FOXE1 in thyroid development has been reported, FOXE1 regulation in other contexts remains unexplored. We pointed to: (i) a role of FOXE1 in controlling the expression of MSX1 and TGF-β3 relevant in craniofacial development and (ii) a causative part of FOXE1 mutations or mice Foxe1(-/-) genotype in the pathogenesis of cleft palate in the Bamforth-Lazarus syndrome. The MSX1 and TGF-β3 up-regulation in response to FOXE1 at both transcriptional and translational levels and the recruitment of FOXE1 to specific binding motifs, together with the transactivation of the promoters of these genes, indicate that MSX1 and TGF-β3 are direct FOXE1 targets. Moreover, we showed that all the known forkhead-domain mutations, but not the polyalanine-stretch polymorphisms, affect the FOXE1 ability to bind to and transactivate MSX1 and TGF-β3 promoters. In 14-day Foxe1(-/-) mice embryos, Tgf-β3 and Msx1 mRNAs were almost absent in palatal shelves compared with Foxe1(+/-) embryos. Our findings give new insights into the genetic mechanisms underlying the Bamforth-Lazarus syndrome-associated facial defects.

Large-sized cartilage constructs suffer from inhomogeneous extracellular matrix deposition due to insufficient nutrient availability. Computational models of nutrient consumption and tissue growth can be utilized as an efficient alternative to experimental trials to optimize the culture of large constructs; models require system-specific growth and consumption parameters. To inform models of the [bovine chondrocyte]-[agarose gel] system, total synthesis rate (matrix accumulation rate+matrix release rate) and matrix retention fractions of glycosaminoglycans (GAG), collagen, and cartilage oligomeric matrix protein (COMP) were measured either in the presence (continuous or transient) or absence of TGF-β3 supplementation. TGF-β3's influences on pyridinoline content and mechanical properties were also measured. Reversible binding kinetic parameters were characterized using computational models. Based on our recent nutrient supplementation work, we measured glucose consumption and critical glucose concentration for tissue growth to computationally simulate the culture of a human patella-sized tissue construct, reproducing the experiment of Hung et al. (2003). Transient TGF-β3 produced the highest GAG synthesis rate, highest GAG retention ratio, and the highest binding affinity; collagen synthesis was elevated in TGF-β3 supplementation groups over control, with the highest binding affinity observed in the transient supplementation group; both COMP synthesis and retention were lower than those for GAG and collagen. These results informed the modeling of GAG deposition within a large patella construct; this computational example was similar to the previous experimental results without further adjustments to modeling parameters. These results suggest that these nutrient consumption and matrix synthesis models are an attractive alternative for optimizing the culture of large-sized constructs.

This work examined the chondrogenic potential of chondrocyte and mesenchymal stem cell (MSC) coculture generated poly(ɛ-caprolactone) (PCL)/extracellular matrix (ECM) hybrid scaffolds. Five different ratios of chondrocytes and MSCs were cocultured to generate cartilage-like ECM within electrospun fibrous scaffolds for 7, 14, and 21 days. These constructs were then devitalized to isolate the chondrogenic effects of the ECM alone. Devitalization was successful at removing cellular matter from the scaffolds, yet did reduce the amount of matrix present in the scaffolds. Following devitalization, the PCL/ECM scaffolds were then cultured with MSCs in serum-free conditions with or without TGF-β3 treatment for 21 days. TGF-β3 supplemented culture caused an induction of chondrogenesis in each scaffold type, but also somewhat masked the subtle differences of the different ECM coatings. Without TGF-β3, the cartilaginous matrix generated by 1:1 cocultures of chondrocytes to MSCs for 14 days supported similar chondrogenic gene expression patterns of MSCs cultured on scaffolds generated by chondrocytes alone. These scaffold formulations had a positive chondrogenic effect on aggrecan, collagen type II, and collagen II/I expression when compared to PCL controls. This study demonstrates that it is possible to utilize cocultures of chondrocytes and MSCs to coat a polymer scaffold with cartilage-like ECM capable of supporting chondrogenic differentiation of MSCs.

Hydrostatic pressure (HP) is a key component of the in vivo joint environment and has been shown to enhance chondrogenesis of stem cells. The objective of this study was to investigate the interaction between HP and TGF-β3 on both the initiation and maintenance of a chondrogenic phenotype for joint tissue derived stem cells. Pellets generated from porcine chondrocytes (CCs), synovial membrane derived stem cells (SDSCs) and infrapatellar fat pad derived stem cells (FPSCs) were subjected to 10 MPa of cyclic HP (4 h/day) and different concentrations of TGF-β3 (0, 1 and 10 ng/mL) for 14 days. CCs and stem cells were observed to respond differentially to both HP and TGF-β3 stimulation. HP in the absence of TGF-β3 did not induce robust chondrogenic differentiation of stem cells. At low concentrations of TGF-β3 (1 ng/mL), HP acted to enhance chondrogenesis of both SDSCs and FPSCs, as evident by a 3-fold increase in Sox9 expression and a significant increase in glycosaminoglycan accumulation. In contrast, HP had no effect on cartilage-specific matrix synthesis at higher concentrations of TGF-β3 (10 ng/mL). Critically, HP appears to play a key role in the maintenance of a chondrogenic phenotype, as evident by a down-regulation of the hypertrophic markers type X collagen and Indian hedgehog in SDSCs irrespective of the cytokine concentration. In the context of stem cell based therapies for cartilage repair, this study demonstrates the importance of considering how joint specific environmental factors interact to regulate not only the initiation of chondrogenesis, but also the development of a stable hyaline-like repair tissue.

The purpose of this study was to examine the role of two of the three transforming growth factor beta (TGF-β) isoforms at the healing tendon-to-bone insertion. The supraspinatus tendons of 64 rats were transected at their bony insertions and repaired to the humeral head. One shoulder of each rat received an osmotic pump for sustained delivery of the following factors at the repair site: (1) TGF-β1 and neutralizing antibodies to TGF-β2 and 3 (TGF-β1 group), (2) TGF-β3 and neutralizing antibodies to TGF-β1 and 2 (TGF-β3 group), (3) neutralizing antibodies to TGF-β1, 2, and 3 (anti-TGF-β group), and (4) saline (saline group). The contralateral shoulders received saline to serve as paired controls. The repairs were evaluated at multiple time points postmortem using histology-based assays and biomechanical testing. Treated shoulders in the TGF-β1 group showed increased type III collagen production compared to the paired control shoulders, indicative of a scar-mediated response. There was a trend toward reduced mechanical properties in the TGF-β1 group, but these changes did not reach statistical significance. The anti-TGF-β group showed no difference in tissue volume, but significantly inferior mechanical properties, compared to the paired control shoulders. The TGF-β3 group did not show any differences compared to the paired control shoulders. Although TGF-β isoforms play important roles in tendon-to-bone development and healing, application of exogenous TGF-β isoforms and neutralizing antibodies to the subacromial space using osmotic pumps did not improve supraspinatus tendon-to-bone healing.

The natural reparative mechanisms triggered by tendon damage often lead to the formation of biomechanically inferior scar tissue that is prone to re-injury. Before the efficient application of stem cell-based regenerative therapies, the processes regulating tenocyte differentiation should first be better understood. Three-dimensional (3D) growth environments under strain and the exogenous addition of transforming growth factor beta3 (TGF-β3) have separately been shown to promote tendon differentiation. The aim of this study was to determine the ability of both of these factors to induce tendon differentiation of equine embryo-derived stem cells (ESCs). ESCs seeded into 3D collagen constructs can contract the matrix to a similar degree to that of tenocyte-seeded constructs and histologically appear nearly identical, with no areas of cartilage or bone tissue deposition. Tendon-associated genes and proteins Tenascin-C, Collagen Type I, and COMP are significantly up-regulated in the 3D ESC constructs compared with tenogenic induction in monolayer ESC cultures. The addition of TGF-β3 to the 3D cultures further up-regulates the expression of these genes and also induces the expression of mature tenocyte markers Tenomodulin and Thrombospondin-4. Our results show that when ESCs are exposed to the intrinsic forces exerted by a 3D culture environment, they express tendon-associated genes and proteins which are indicative of tenocyte lineage differentiation and that this effect is synergistically enhanced and accelerated by the addition of TGF-β3.

Patients with diabetes mellitus (DM) may develop corneal complications and delayed wound healing. The aims of this study are to characterize the molecular signatures and biological pathways leading to delayed epithelial wound healing and to delineate the involvement of TGFβ3 therein. Genome-wide cDNA microarray analysis revealed 1,888 differentially expressed genes in the healing epithelia of normal (NL) versus type 1 DM rat corneas. Gene ontology and enrichment analyses indicated TGFβ signaling as a major altered pathway. Among three TGFβ isoforms, TGF-β1 and β3 were upregulated in response to wounding in NL corneal epithelial cells (CECs), whereas the latter was greatly suppressed by hyperglycemia in rat type 1 and 2 and mouse type 1 DM models. Functional analysis indicated that TGF-β3 contributed to wound healing in NL corneas. Moreover, exogenously added TGF-β3 accelerated epithelial wound closure in type 2 rat and type 1 mouse DM corneas via Smad and PI3K-AKT signaling pathways, autoregulation, and/or upregulation of Serpine1, a well-known TGFβ target gene. Taken together, our study for the first time provides a comprehensive list of genes differentially expressed in the healing CECs of NL versus diabetic corneas and suggests the therapeutic potential of TGF-β3 for treating corneal and skin wounds in diabetic patients.

Following injury, skeletal muscle achieves repair by a highly coordinated, dynamic process resulting from interplay among numerous inflammatory, growth factors and myogenic regulators. To identify genes involved in muscle regeneration, we used a microarray analysis; there was a significant increase in the expression of a group of integrin genes. To verify these results, we used RT-PCR and Western blotting and found that 12 integrins were up-regulated from 3 h to 15 days following injury. Following muscle injury, integrin-β3 was initially expressed, mainly in macrophages. In integrin-β3 global KO mice, the expression of myogenic genes was decreased and muscle regeneration was impaired, whereas fibrosis was enhanced versus events in wild type (WT) mice. The mechanism for these responses in integrin-β3 KO mice included an infiltration of macrophages that were polarized into the M2 phenotype. These macrophages produced more TGF-β1 and increased TGF-β1/Smad signaling. In vitro, we confirmed that M2 macrophages lacking integrin-β3 produced more TGF-β1. Furthermore, transplantation of bone marrow cells from integrin-β3 KO mice into WT mice led to suppression of the infiltration and accumulation of macrophages into injured muscles. There was also impaired muscle regeneration with an increase in muscle fibrosis. Our results demonstrate that integrin-β3 plays a fundamental role in muscle regeneration through a regulation of macrophage infiltration and polarization leading to suppressed TGF-β1 production. This promotes efficient muscle regeneration. Thus, an improvement in integrin-β3 function could stimulate muscle regeneration.