BACKGROUND

Animal studies report photodynamic therapy (PDT) to improve healing of excisional wounds; the mechanism is uncertain and equivalent human studies are lacking.

OBJECTIVE

To explore the impact of methyl aminolaevulinate (MAL)-PDT on clinical and microscopic parameters of human cutaneous excisional wound healing, examining potential modulation through production of transforming growth factor (TGF)-β isoforms.

METHODS

In 27 healthy older men (60-77 years), a 4-mm punch biopsy wound was created in skin of the upper inner arm and treated with MAL-PDT three times over 5 days. An identical control wound to the contralateral arm was untreated and both wounds left to heal by secondary intention. Wounds were re-excised during the inflammatory phase (7 days, n = 10), matrix remodelling (3 weeks, n = 8) and cosmetic outcome/dermal structure (9 months, n = 9). Production of TGF-β1, TGF-β3 and matrix metalloproteinases (MMPs) was assessed by immunohistochemistry alongside microscopic measurement of wound size/area and clinical assessment of wound appearance.

RESULTS

MAL-PDT delayed re-epithelialization at 7 days, associated with increased inflammation. However, 3 weeks postwounding, treated wounds were smaller with higher production of MMP-1 (P = 0·01), MMP-9 (P = 0·04) and TGF-β3 (P = 0·03). TGF-β1 was lower than control at 7 days and higher at 3 weeks (both P = 0·03). At 9 months, MAL-PDT-treated wounds showed greater, more ordered deposition of collagen I, collagen III and elastin (all P < 0·05).

CONCLUSIONS

MAL-PDT increases MMP-1, MMP-9 and TGF-β3 production during matrix remodelling, ultimately producing scars with improved dermal matrix architecture.

UNASSIGNED

We have identified a gene YOD1 encoding deubiquitinating enzyme (DUB) responsible for nonsyndromic cleft lip with or without cleft palate (NSCL/P). We aimed to determine the effects of YOD1 RNA interference (RNAi) on cell proliferation and migration, playing an important role in lip and palate formation, and to clarify whether the mechanisms involved TGF-β3 signaling associated with NSCL/P.

UNASSIGNED

RNAi was applied to construct vectors expressing YOD1 small interference RNAs (siRNAs). The vectors were transfected into the human oral keratinocytes (HOK) cells. The cell proliferation and migration were evaluated by the cell counting kit-8 (CCK-8) assay and wound healing assay, respectively. The mRNA levels were detected by quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR). The protein levels were investigated by western blotting.

UNASSIGNED

The proliferation of YOD1 siRNA-transfected HOK cells was remarkably inhibited. The migration rate was significantly decreased in the YOD1 siRNA-transfected HOK cells. The TGF-β3 mRNA and protein levels were decreased significantly by siRNA-mediated knockdown of YOD1. YOD1 RNAi reduced the phosphor-Smad2/3 levels significantly.

UNASSIGNED

YOD1 RNAi may inhibit cell proliferation and migration associated with the pathogenesis of NSCL/P through TGF-β3 signaling. The study indicates a novel role of YOD1 in regulating TGF-β3 signaling to affect cell proliferation and migration resulting in NSCL/P.

To study the expression profile of bone morphogenetic proteins and transforming growth factor-βs (BMPs and TGFβs), coral-derived calcium carbonate-based macroporous bioreactors with limited conversion to hydroxyapatite (7% HA/CC) were pre-loaded with and without 250 μg hTGF-β3 and implanted in the rectus abdominis of 3 non-human primates Papio ursinus euthanized on day 60. To investigate the required dose of hNoggin, a BMPs antagonist that controls the induction of bone formation, 7% HA/CC were pre-loaded with 150 μg hNoggin, with 125 μg hTGF-β3/150 μg hNoggin, with or without 125 μg hTGF-β3 and implanted in the r. abdominis of 3 additional animals euthanized on day 90. Quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) evaluated the expression' profile of BMP-2, BMP-3, BMP-4, BMP-6, BMP-7 and TGF-β1, -β2, and -β3 in tissue generating bioreactors as well as in the adjacent r. abdominis muscle. On day 60, 250 μg hTGF-β3 induced bone formation at the periphery of the implanted bioreactors only. On day 90, 125 μg hTGF-β3/treated bioreactors showed the induction of bone formation throughout the macroporous spaces. Untreated bioreactors induced bone, 4.11% vs. 2.00% on days 60 and 90, respectively. In hTGF-β3/treated bioreactors, BMP-2 and BMP-3 were up-regulated at both time periods, both in the homogenized constructs and in the adjacent r. abdominis muscle whilst BMP-4 in the homogenized construct only. In untreated 7% HA/CC constructs, BMP-2 was up-regulated in the macroporous construct only. On day 60, 250 μg hTGF-β3/treated and untreated macroporous constructs showed up-regulation of TGF-β1 with a six fold increase vs. TGF-β1 expression in adjacent muscle of untreated constructs. TGF-β2 was down regulated in both untreated and 250 μg hTGF-β3/treated bioreactors. On day 60, 250 μg hTGF-β3/treated bioreactors showed TGF-β3 expression in untreated, treated and adjacent muscle tissues. On day 90, BMP-2 was up-regulated, with BMP-3 up-regulation both with and without hNoggin. BMP-4 expression was reduced, with down-regulation of BMP-6 and BMP-7 with and without hNoggin. BMPs expression is finely tuned by hNoggin that blocks BMPs resulting in minimal if any induction of bone formation. The primary differentiating events in untreated bioreactors develop within the macroporous spaces after stem cells invasion and differentiation with lack of BMP-2 expression in the adjacent muscle. In hTGF-β3/treated bioreactors, both the adjacent muscle and the macroporous bioreactor show BMP-2 up-regulation, correlating with the temporo/spatial rapid induction of bone formation at the periphery of the bioreactors only. The latter may be the result of the initiation of a sequential chain of cellular induction rapidly recruiting pericytic perivascular myoblastic cells adjacent to the implanted bioreactors directly transformed into secreting osteoblasts.

OBJECTIVE

This study aims to deliver both transforming growth factor β3 (TGF-β3) and shRNA targeting type I collagen (Col I) by optimal construction and application of various dual-functioning lentiviral vectors to induce Col I-suppressed chondrogenesis in synovium-derived mesenchymal stem cells (SMSCs).

METHODS

We constructed four lentiviral vectors (LV-1, LV-2, LV-3 and LV-4) with various arrangements of the two expression cassettes in different positions and orientations. Col I inhibition efficiency and chondrogenic markers were assessed with qPCR, ELISA and staining techniques. Among the four vectors, LV-1 has two distant and reversely oriented cassettes, LV-2 has two distant and same-oriented cassettes, LV-3 has two proximal and reversely oriented cassettes, and LV-4 has two proximal and same-oriented cassettes. Col I and chondrogenic markers, including type II collagen (Col II), aggrecan and glycosaminoglycan (GAG), were examined in SMSCs cultured in 3-D alginate hydrogel.

RESULTS

All of the four vectors showed distinct effects in Col I level as well as diverse inductive efficiencies in upregulation of the cartilaginous markers. Based on real-time PCR results, LV-1 was optimal towards Col I-suppressed chondrogenesis.

CONCLUSIONS

LV-1 vector is competent to promote Col I-suppressed chondrogenesis in SMSCs.

The CCN family of proteins plays important roles in development and homeostasis of bone and cartilage. To understand the role of CCN4 in chondrogenesis, human bone marrow stromal cells (hBMSCs) were transduced with CCN4 adenovirus (adCCN4) or siRNA to CCN4 (siCCN4) in the presence or absence of transforming growth factor-β3 (TGF-β3). Overexpression of CCN4 enhanced TGF-β3-induced SMAD2/3 phosphorylation and chondrogenesis of hBMSCs in an in vitro assay using a micromass culture model. On the other hand, knockdown of CCN4 inhibited the TGF-β3-induced SMAD2/3 phosphorylation and synthesis of cartilage matrix in micromass cultures of hBMSCs. Immunoprecipitation-western blot analysis revealed that CCN4 bound to TGF-β3 and regulated the ability of TGF-β3 to bind to hBMSCs. In vivo analysis confirmed there was a significant decrease in the gene expression levels of chondrocyte markers in cartilage samples from Ccn4-knock out (KO) mice, compared to those from wild type (WT) control. In order to investigate the regenerative properties of the articular cartilage in Ccn4-KO mice, articular cartilage defects were surgically performed in the knee joints of young mice, and the results showed that the cartilage was partially repaired in WT mice, but not in Ccn4-KO mice. In conclusion, these results show, for the first time, that CCN4 has a positive influence on chondrogenic differentiation by modulating the effects of TGF-β3.

BACKGROUND

It is increasingly recognized that exposure to ambient fine particles (PM(2.5)) is a risk factor for the development of cardiovascular events. This study was to explore the link between PM(2.5) exposure and viral myocarditis in the functional mechanism of Th17 cells.

METHODS

Male BALB/c mice were administered an intratracheal (i.t.) instillation of 10 mg/kg b.w. PM(2.5) particles. Twenty-four hours later, the mice were injected intraperitoneally (i.p.) with 100 μl of coxsackievirus B3 (CVB3) diluted in Eagle's minimal essential medium (EMEM). Seven days after the treatment, pulmonary and cardiac tissues were examined.

RESULTS

The results showed that preexposure to PM(2.5) increased the cardiac and pulmonary injuries and viral replication in the heart of CVB3-infected mice along with an increase in CD4(+) IL-17(+) cells in the spleen and heart. The mRNA expressions of interleukin-17A (IL-17A), perforin, transforming growth factor-β (TGF-β) and RORγt were up-regulated in PM(2.5)-pretreated mice than that in the virus-treated mice. Additionally, compared to virus-treated mice, the cardiac protein expressions of IL-17A and matrix metalloproteinases-2 (MMP-2) were increased, but interferon-γ (IFN-γ) and metalloproteinases-1 (TIMP-1) were decreased in PM(2.5)-pretreated mice. Interestingly, PM(2.5) caused IFN-γ decreased, whereas CVB3 caused a dramatic increase in IFN-γ. Subsequently, preexposure to PM(2.5) induced a slight increase of IFN-γ in the sera of CVB3-infected mice.

CONCLUSIONS

These results demonstrated that PM(2.5) exposure exacerbated virus-induced myocarditis possibly through the increase in Th17-mediated viral replication, perforin response and imbalance of MMP-2/TIMP-1. These findings provided supportive evidence for the epidemiological research that ambient particles could increase the occurrence and development of cardiovascular diseases.

The objectives of this study were to determine whether Staphylococcus aureus chronic intramammary infection (IMI) influences protein expression of TGF-β subfamily components and collagen I and to examine the histomorphometric changes that occur in mammary stroma and parenchyma during active mammary gland involution. Twenty-one Holstein non-pregnant cows in late lactation either uninfected or with chronic natural S. aureus IMI were included in this study. Cows were slaughtered at 7, 14 and 21d after cessation of milking and samples for immunohistochemical and morphometric analysis were taken. Protein expression of TGF-β1, TGF-β2 and TGF-β3 was significantly higher in chronically infected quarters than in uninfected controls at the three involution stages studied. Immunostaining of TGF-βR1 and TGF-βR3 and collagen I was significantly higher in S. aureus-infected quarters than in uninfected controls at every involution time evaluated. The percentages of tissue area composed of parenchyma and intralobular stroma were significantly higher in S. aureus-infected than in uninfected quarters. Chronic S. aureus mastitis modifies protein expression of the three TGF-β isoforms and type 1 and 3 receptors, which was associated with changes directed to limit the scope of inflammation and injury to the host.

Intervertebral disc (IVD) degeneration is associated with several pathophysiologic changes of the IVD, including dehydration of the nucleus pulposus (NP). Tissue engineering strategies may be used to restore both biological and mechanical function of the IVD following removal of NP tissue during surgical intervention. Recently, photocrosslinked carboxymethylcellulose (CMC) hydrogels were shown to support chondrogenic, NP-like extracellular matrix (ECM) elaboration by human mesenchymal stromal cells (hMSCs) when supplemented with TGF-β3; however, mechanical properties of these constructs did not reach native values. Fabrication parameters (i.e., composition, crosslinking density) can influence the bulk mechanical properties of hydrogel scaffolds, as well as cellular behavior and differentiation patterns. The objective of this study was to evaluate the influence of CMC macromer concentration (1.5, 2.5 and 3.5 % weight/volume) on bulk hydrogel properties and NP-like matrix elaboration by hMSCs. The lowest macromer concentration of 1.5 % exhibited the highest gene expression levels of aggrecan and collagen II at day 7, corresponding with the largest accumulation of glycosaminoglycans and collagen II by day 42. The ECM elaboration in the 1.5 % constructs was more homogeneously distributed compared to primarily pericellular localization in 3.5 % gels. The 1.5 % gels also displayed significant improvements in mechanical functionality by day 42 compared to earlier time points, which was not seen in the other groups. The effects of macromer concentration on matrix accumulation and organization are likely attributed to quantifiable differences in polymer crosslinking density and diffusive properties between the various hydrogel formulations. Taken together, these results demonstrate that macromer concentration of CMC hydrogels can direct hMSC matrix elaboration, such that a lower polymer concentration allows for greater NP-like ECM assembly and improvement of mechanical properties over time.

OBJECTIVE

During in vitro chondrogenesis of human mesenchymal stem cells (hMSCs) hypertrophy is an inadvertent event associated with cell differentiation toward the osteogenic lineage. Up to now, there is no stringent experimental control mechanism to prevent hypertrophy of MSCs. Microgravity is known to have an impact on osteogenesis. In this study, the influence of simulated microgravity (SMG) on both chondrogenesis and hypertrophy of hMSCs was evaluated.

METHODS

A bioreactor using a rotating wall vessel was constructed to simulate microgravity. Pellet cultures formed from hMSCs (P5) were supplemented with human transforming growth factor-β3 (TGF-β3). The hMSC pellet cultures treated with TGF-β3 were either kept in SMG or in a control system. After three weeks of culture, the chondrogenic differentiation status and level of hypertrophy were examined by safranin-O staining, immunohistochemistry and quantitative real-time PCR.

RESULTS

SMG reduced the staining for safranin-O and collagen type II. The expression of collagen type X α1 chain (COL10A1) and collagen type II α1 chain (COL2A1) were both significantly reduced. There was a higher decrease in COL2A1 than in COL10A1 expression, resulting in a low COL2A1/COL10A1 ratio.

CONCLUSIONS

SMG reduced hypertrophy of hMSCs during chondrogenic differentiation. However, the expression of COL2A1 was likewise reduced. Even more, the COL2A1/COL10A1 ratio decreased under SMG conditions. We therefore assume that SMG has a significant impact on the chondrogenic differentiation of hMSCs. However, due to the high COL2A1 suppression under SMG, this culture system does not yet seem to be suitable for a potential application in cartilage repair.

A number of growth factors determine the proliferation of myoblasts and therefore the number of ultimate myofibers. The members of transforming growth factor-beta (TGF-β) family and the fibroblast growth factor 2 (FGF2) have profound effects on skeletal myoblasts proliferation in various animal systems. To investigate their involvement in different stages of avian skeletal muscle development in vivo, we detected the mRNA expression and DNA methylation profiles of TGF-β2, TGF-β3, FGF2 and their downstream mediators in leg muscles at embryonic day 10, day of hatch and day 45 posthatch, using both Arbor Acres meat-type and White Leghorn egg-type chickens. By real-time PCR, we found that the expression levels of TGF-β2, TGF-β3, Smad3 and FGF2 were significantly (P≤0.01) higher at embryonic day 10, a developmental window of abundant fetal myoblasts expansion, by comparison to day of hatch and day 45 posthatch. The methylation status of the 5' end region of these four genes was examined subsequently. A section of a CpG island in the 5' end region of FGF2 was significantly hypomethylated (P≤0.01) at embryonic day 10, compared with neonatal and postnatal stages in both stocks. Our results suggested that TGF-β2, TGF-β3, Smad3 and FGF2 may play important roles in fetal myoblasts proliferation in chicken hindlimb, and the transcription of FGF2 in this wave of myogenesis could be affected by DNA methylation in 5' flanking region. These outcomes contribute to our knowledge of the growth factors in avian myogenesis. Further investigation is needed to confirm and fully understand their functions in fetal limb myogenesis in birds.

BACKGROUND

The aim of this study was to compare the effects of preoperative subcutaneous platelet-rich plasma and fibrin glue administration on skin flap survival.

METHODS

One week before surgery; saline, platelet-rich plasma, fibrin glue, and thrombin solutions were applied under rat skin flap areas in Groups I, II, III, and IV, respectively. Unipedicled epigastric flaps were elevated in the first three groups but could not be elevated in Group IV because of preoperative abdominal skin necrosis. Necrotic area measurements, microangiography, and histological and immunohistochemical evaluations were performed.

RESULTS

Platelet-rich plasma reduced the percentage of necrotic area when compared to other groups. Histologically and microangiographically an increased number of arterioles were observed in the platelet-rich plasma group. Thrombin (when used alone) caused abdominal skin necrosis. Increased expression of VEGF and PDGF was found in all platelet-rich plasma-treated flaps. There was no significant difference between groups with respect to TGF-β3 staining intensity.

CONCLUSIONS

In this study preoperative administration of platelet-rich plasma mimicked the pharmacological delay effect and enhanced flap survival. Individual use of thrombin was found to be unsuitable in flap surgery.

METHODS

This journal requires that authors assign a level of evidence to each article.

Despite an increased interest in the use of hydrogel encapsulation and cellular self-assembly (often termed "self-aggregating" or "scaffold-free" approaches) for tissue-engineering applications, to the best of our knowledge, no study to date has been undertaken to directly compare both approaches for generating functional cartilaginous grafts. The objective of this study was to directly compare self-assembly (SA) and agarose hydrogel encapsulation (AE) as a means to engineer such grafts using passaged chondrocytes. Agarose hydrogels (5 mm diameter × 1.5 mm thick) were seeded with chondrocytes at two cell seeding densities (900,000 cells or 4 million cells in total per hydrogel), while SA constructs were generated by adding the same number of cells to custom-made molds. Constructs were either supplemented with transforming growth factor (TGF)-β3 for 6 weeks, or only supplemented with TGF-β3 for the first 2 weeks of the 6 week culture period. The SA method was only capable of generating geometrically uniform cartilaginous tissues at high seeding densities (4 million cells). At these high seeding densities, we observed that total sulphated glycosaminoglycan (sGAG) and collagen synthesis was greater with AE than SA, with higher sGAG retention also observed in AE constructs. When normalized to wet weight, however, SA constructs exhibited significantly higher levels of collagen accumulation compared with agarose hydrogels. Furthermore, it was possible to engineer such functionality into these tissues in a shorter timeframe using the SA approach compared with AE. Therefore, while large numbers of chondrocytes are required to engineer cartilaginous grafts using the SA approach, it would appear to lead to the faster generation of a more hyaline-like tissue, with a tissue architecture and a ratio of collagen to sGAG content more closely resembling native articular cartilage.

It is well known that terminally differentiated cells derived from mesenchymal stem cells (MSCs) will lose the immunomodulation capacity. NANOG is known to be a core transcription factor in the maintenance of stem cell specific features or stemness. To evaluate whether NANOG was involved in the immunomodulation effects of MSCs, MSCs' immunomodulation capacity on lymphocyte activation and proliferation before or after endogenous NANOG interference was investigated. We found that MSCs' inhibitory effects on lymphocyte activation and proliferation was significantly weakened after NANOG knockdown. In addition, NANOG RNAi and chromatin immunoprecipitation experiments showed that NANOG suppressed the expression and secretion of DKK-1, transforming growth factor-beta1 (TGF-β1), TGF-β2, and TGF-β3, which are all important factors mediating MSCs' immunomodulation capacity. Based on these data, we propose that NANOG plays an important role in maintaining the immunomodulation functions of MSCs by regulating the expression and secretion of TGF-β1, TGF-β2, TGF-β3, and DKK-1.

Transforming growth factor beta isoforms (TGF-β1, -β2, and -β3) are cytokines associated with a wide range of biological processes in oncology including tumor cell invasion and migration, angiogenesis, immunosuppression, as well as regulation of tumor stem cell properties. Hence, blocking the TGF-β signaling pathways may have a multifold therapeutic benefit for the treatment of solid tumors. Here, we describe the identification and selection processes for the development of highly potent and selective chemically modified antisense oligodeoxynucleotides (fully phosphorothioate locked nucleic acid gapmers) allowing effective and selective suppression of TGF-β isoform expression in cell-based assays and in vivo preclinical models.

OBJECTIVE

To investigate the mechanisms inducing and maintaining the permanent elimination of low dose hyper-radiosensitivity (HRS) in cells given a dose of 0.3 Gy at low dose-rate (LDR) (0.3 Gy/h).

METHODS

Two human HRS-positive cell lines (T-47D, T98G) were used. The effects of pretreatments with transforming growth factor beta (TGF-β) neutralizers, TGF-β3 or peroxynitrite scavenger on HRS were investigated using the colony assay. Cytoplasmic levels of TGF-β3 were measured using post-embedding immunogold electron microscopic analysis.

RESULTS

TGF-β3 neutralizer inhibited the removal of HRS by LDR irradiation. Adding 0.001 ng/ml TGF-β3 to cells removed HRS in T98G cells while 0.01 ng/ml additionally induced resistance to higher doses. Cytoplasmic levels of TGF-β3 were higher in LDR-primed cells than in unirradiated cells. The presence of the peroxynitrite scavenger uric acid inhibited the effect of LDR irradiation. Furthermore, the permanent elimination of HRS in LDR-primed cells was reversed by treatment with uric acid. The removal of HRS by medium from hypoxic cells was inhibited by adding TGF-β3 neutralizer to the medium before transfer or by adding hypoxia inducible factor 1 (HIF-1) inhibitor chetomin to the cell medium during hypoxia.

CONCLUSIONS

TGF-β3 is involved in the regulation of cellular responses to small doses of acute irradiation. TGF-β3 activation seems to be induced by low dose-rate irradiation by a mechanism involving inducible nitric oxide (iNOS) and peroxynitrite, or during cycling hypoxia by a mechanism most likely involving HIF-1. The study suggests methods to turn resistance to doses in the HRS-range on (by TGF-β3) or off (by TGF-β3 neutralizer or by peroxynitrite inhibition).

The pathophysiology of human keratoconus (KC), a bilateral progressive corneal disease leading to protrusion of the cornea, stromal thinning, and scarring, is not well-understood. In this study, we investigated a novel sphingolipid (SPL) signaling pathway through which KC may be regulated. Using human corneal fibroblasts (HCFs) and human KC cells (HKCs), we examined the SPL pathway modulation. Both cell types were stimulated by the three transforming growth factor (TGF)-β isoforms: TGF-β1 (T1), TGF-β2 (T2), and TGF-β3 (T3). All samples were analyzed using lipidomics and real-time PCR. Our data showed that HKCs have increased levels of signaling SPLs, ceramide (Cer), and sphingosine 1-phosphate (S1P). Treatment with T1 reversed the increase in Cer in HKCs and treatment with T3 reversed the increase in S1P. S1P3 receptor mRNA levels were also significantly upregulated in HKCs, but were reduced to normal levels following T3 treatment. Furthermore, stimulation with Cer and S1P led to significant upregulation of fibrotic markers in HCFs, but not in HKCs. Additionally, stimulation with a Cer synthesis inhibitor (FTY720) led to significant downregulation of specific fibrotic markers in HKCs (TGF-β1, collagen type III, and α smooth muscle actin) without an effect on healthy HCFs, suggesting a causative role of Cer and S1P in fibrogenesis. Overall, this study suggests an association of the SPL signaling pathway in KC disease and its relation with the TGF-β pathway.

Controversy still exists regarding the role of the TGF-β in neovascular age-related macular degeneration (nAMD), a major cause of severe visual loss in the elderly in developed countries. Here, we measured the concentrations of active TGF-β1, TGF-β2, and TGF-β3 by ELISA in the aqueous humor of 20 patients affected by nAMD, who received 3 consecutive monthly intravitreal injections of anti-VEGF-A antibody. Samples were collected at baseline (before the first injection), month 1 (before the second injection), and month 2 (before the third injection). The same samples were used in a luciferase-based reporter assay to test the TGF-β pathway activation. Active TGF-β1 concentrations in the aqueous humor were below the minimum detectable dose. Active TGF-β2 concentrations were significantly lower at baseline and at month 1, compared to controls. No significant differences in active TGF-β3 concentration were found among the sample groups. Moreover, TGF-β pathway activation was significantly lower at baseline compared to controls. Our data corroborate an anti-angiogenic role for TGF-β2 in nAMD. This should be considered from the perspective of a therapy using TGF-β inhibitors.

Gene delivery takes advantage of cellular mechanisms to express gene products and is an efficient way to deliver them into cells, influencing cellular behaviours and expression patterns. Among the delivery methods, viral vectors are applied due to their high efficiency. Two typical viral vectors for gene delivery include lentiviral vector for integrative transduction and adenoviral vector for transient episomal transduction, respectively. The selection and formulation of proper viral vectors applied to cells can modulate gene expression profiles and further impact the downstream pathways. In this study, recombinant lentiviral and adenoviral vectors were co-transduced in a synovial mesenchymal stem cells (SMSCs)-based articular chondrogenic system by which two transgenes were co-delivered - the gene for transforming growth factor (TGF)β3, to facilitate SMSC chondrogenesis, and the gene for small hairpin RNA (shRNA), targeting the mRNA of type I collagen (Col I) α1 chain to silence Col I expression and minimize fibrocartilage formation. Delivery of either gene could be achieved with either lentiviral or adenoviral vectors. Therefore, co-delivery of the two transgenes via the two types of vectors was performed to determine which combination was optimal for three-dimensional (3D) articular chondrogenesis to construct articular hyaline cartilage tissue. Suppression of Col I and expression of cartilage markers, including type II collagen, aggrecan and cartilage oligomeric matrix protein (COMP), were assessed at both the transcriptome and protein phenotypic levels. It was concluded that the combination of lentiviral-mediated TGFβ3 release and adenoviral-mediated shRNA expression (LV-T + Ad-sh) generally demonstrated optimal efficacy in engineered articular cartilage with SMSCs.

Transforming growth factors-beta (TGF-b1, -b2, and -b3) is secreted from osteoblasts and act as an anabolic factor through its influence of osteoblast proliferation and differentiation and on the synthesis of bone matrix. The ability of progestins to promote bone formation is attributed to their ability to act upon osteoblasts. In the present studies, we observed the effects of progesterone on three TGF-b isoforms (b1, b2, and b3) expression in culture of normal human osteoblast-like cells (hOB). mRNA levels were determined by Northern blot analysis, and TGF-b1, -b2, and -b3 concentrations in conditioned media were determined by ELISA, After 12-24 hours of treatment, progesterone at 10-9 M increased TGF-b1, -b2, and -b3 mRNA levels and protein production in cultures of hOB, treatment with increasing dose of progesterone caused a dose-dependent increase in the expression of TGF-b1, -b2, and -b3 mRNA and protein by hOB. The data suggest that a direct action of progesterone on osteoblasts may be mediated by up-regulated TGF-b isoforms production.

Decoy receptor 3 (DcR3), a novel member of the tumor necrosis factor receptor (TNFR) family, was recently reported to be associated with tumorigenesis and metastasis. However, the role of DcR3 in human colorectal cancer (CRC) has not been fully elucidated. In this study, we found that DcR3 expression was significantly higher in human colorectal cancer tissues than in paired normal tissues, and that DcR3 expression was strongly correlated with tumor invasion, lymph node metastases and poor prognoses. Moreover, DcR3 overexpression significantly enhanced CRC cell proliferation and migration in vitro and tumorigenesis in vivo. Conversely, DcR3 knockdown significantly repressed CRC cell proliferation and migration in vitro, and DcR3 deficiency also attenuated CRC tumorigenesis and metastasis in vivo. Functionally, DcR3 was essential for TGF-β3/SMAD-mediated epithelial-mesenchymal transition (EMT) of CRC cells. Importantly, cooperation between DcR3 and TGF-β3/SMAD-EMT signaling-related protein expression was correlated with survival and survival time in CRC patients. In conclusion, our results demonstrate that DcR3 may be a prognostic biomarker for CRC and that this receptor facilitates CRC development and metastasis by participating in TGF-β3/SMAD-mediated EMT of CRC cells.

The aim of the study was to determine temporal TGFB1, TGFB2 and TGFB3 gene expression profiles in the anterior lens capsule of paediatric patients with posttraumatic cataract. The patient group comprised 22 children selected with a fragment of anterior lens capsule obtained during elective cataract surgery and sampled for molecular analysis. The levels of TGF-β isoforms in the anterior lens capsule were determined based on the number of mRNA copies per 1 μg total RNA by real-time qRTPCR. Three time-related result clusters were identified based on hierarchical cluster analysis: 2.2, 4.4 and 15.0 months (time span from injury to anterior capsule sampling during surgery) and compared with regard to temporal gene expression profile and quantitative relations of TGF-β1, 2 and 3 mRNAs. TGF-β1, TGF-β2, and TGF-β3 mRNAs were detected in all anterior lens capsule samples. A comparative analysis revealed: TGF-β1>TGF-β2>TGF-β3 during the entire observation period. The TGF-β mRNA levels continued to increase up to four months after injury, then returning close to the base levels after around 15 months. The expression patterns of TGF-β isoforms showed a similar tendency. Differences in the expression levels of TGF-β1 and TGF-β2 between the particular clusters were statistically significant. Posttraumatic transcriptional activities of TGF-β1 and TGF-β2 in the anterior lens capsule of paediatric patients depend on the time elapsing from injury. Our findings indicate that the transcriptional activities of TGFB family genes show a transient period of over-expression during the months after injury. TGF-β1 is a dominant isoform expressed in lens epithelial cells following injury.

BACKGROUND

Although various alterative models of therapy are used for cartilage repair, no definite conclusion has been reached. Glucosamine (GlcN) is widely used as a nutritional supplement. However, the clinical- evidence-based outcome of GlcN administration remains controversial. N-acetyl-D-glucosamine (GlcNAc), a derivative of GlcN, shows chondroprotective activity and mediates the activation of articular chondrocytes. Therefore, we investigated the effect of intra-articular administration of GlcNAc in rabbits' knee joints with experimental full-thickness articular cartilage (FTAC) defects.

METHODS

Twelve male adult New Zealand white rabbits, providing 24 knees, were used in this study. FTAC defects were created in the high-weight-bearing area of the medial femoral condyles of bilateral knees. All rabbits were randomly allocated to analysis at postsurgical week 4 or postsurgical week 12. In the week 4 group, rabbits' knees (six per group) were intra-articularly injected with normal saline or with GlcNAc twice per week for 3 weeks, beginning 1 week postoperatively. In the week 12 group, the rabbits' knees (six in each group) were intra-articularly injected with normal saline or with GlcNAc twice per week for 4 weeks, beginning 1 week postoperatively. Rabbits were sacrificed at 4 or 12 weeks after surgery for macroscopic, histological and radiological examinations of the knee joints.

RESULTS

All rabbits had no systemic or local adverse effects. The saline and GlcNAc groups showed visible differences in healing of the FTAC defect at the end of testing. At week 4, the GlcNAc group had a higher level of collagen type II (COL II) and showed up-regulated production of transforming growth factor (TGF)-β2 and TGF-β3, suggesting the involvement of endogenous growth factors. At week 12, the GlcNAc group displayed formation of hyaline-like cartilage regeneration with mature chondrocytes (SOX9+), robust glycosaminoglycan (GAG) content, and positive COL II content in both the adjacent cartilage and reparative sites. However, the saline group demonstrated mainly fibrocartilage scar tissue, indicating COL I expression. Furthermore, the GlcNAc group had significantly higher bone volume per tissue volume and higher trabecular thickness than the saline group.

CONCLUSIONS

Intra-articular GlcNAc may promote the repair of experimental FTAC defects in the rabbit knee joint model.

The incidence of heart failure in the population increases steeply among older people. Overactivation of the sympathetic nervous system is associated to and responsible for worsening of heart failure. This study describes the influence of aging on short-term left ventricular (LV) adaptation to b-adrenergic stimulation in Wistar rats. In controls at 18 mo, interstitial fibrosis was increased with respect to 3-mo-old rats, whereas myocytes dimension and the messenger RNA (mRNA) abundance of atrial natriuretic peptide (ANP), a2(I) procollagen, transforming growth factor (TGF-b1, TGF-b3), and secreted protein, acidic and rich in cysteine (SPARC) were not different. To determine how aging affects LV adaptation to adrenergic stimulation, two groups of animals received isoproterenol (ISO, 1 mg/kg/d) for 3 days. There was no significant difference between young and older rats with respect to increase in LV weight, myocytes dimension, and mRNA abundance of all the genes considered, except a1(III) procollagen. These findings indicate that despite limited compensatory hypertrophy and higher fibrosis, LV from aged nonsenescent rats preserves the capacity to adapt to b-adrenergic stimulation through the upregulation of several genes encoding extracellular matrix-related proteins.

Myocardial fibrosis is closely associated with heart failure because myocardial fibrosis may cause the loss of normal cardiac function. Endoglin is a homeodimeric membrane glycoprotein, a co-receptor of transforming growth factor-β1 (TGF-β1) and β3. Endoglin is a potent mediator of profibrotic effects of angiotensin II on cardiac fibroblasts and can modulate the effect of TGF-β1 on extracellular matrix synthesis. These data indicate that endoglin plays an important role in fibrogenesis in cardiac remodeling. Endoglin induced by TGF-β1 is largely through PI-3 kinase, Akt, Smad3/4 and endoglin promoter pathways. Endoglin was upregulated in pressure- overload, volume-overload heart failure and acute myocardial infarction and was associated with myocardial fibrosis. Silencing endoglin expression could attenuate myocardial fibrosis and improve survival in animal study. Endoglin expression was increased in failing left ventricle before use of left ventricle assist device, and reduced back to control levels after use of left ventricle assist device. Targeting endoglin may provide a potentially unique and novel therapeutic approach for reducing myocardial fibrosis in patients with heart failure.