Fetal wound healing occurs rapidly and without scar formation early in gestation. Studying the mechanisms of scarless repair can lead to novel scar-preventive approaches. In fetal wounds, collagen is deposited early and is fine and reticular with less cross-linking. Several important differences of fetal vs. postgestational wound-healing response have been determined, such as the presence of less inflammation, higher hyaluronic acid concentration and a greater ratio of collagen type III to type I. Compared with typical wounds, there are also altered ratios of signaling molecules, such as higher ratios of transforming growth factor (TGF)-β3 to TGF-β1 and -β2, and matrix metalloproteinases to tissue inhibitors of metalloproteinases. Furthermore, fetal fibroblasts do not exhibit TGF-β1-induced collagen production compared with their mature counterparts. Patterning genes (homeobox genes) involved in organogenesis are more active in the fetal period and are believed to be the "first domino" in the fetal cutaneous wound repair regulatory cascade. The recommended scar-preventive agents, such as Scarguard MD®, silicone gel and sheet, Seprafilm® Bioresorbable Membrane, topical hyaluronan, onion extract, oral tamoxifen and 585-nm pulsed dye laser are reviewed in this study. Despite the lack of supporting evidence, there is a widespread false presumption that the acceleration of healing with the widely assumed scar-preventive commercial agents is associated with decreased scar formation. Humans are erroneously inclined to make a negative correlation between the healing rate and the degree of scar formation, while such a correlation does not exist in reality. Despite the importance of scar prevention, no FDA-approved therapy for this purpose is available in the 21st century, which reflects the important challenges, such as the presence of redundant pathways, that these approaches are facing.

The cervix is central to the female genital tract immune response to pathogens and foreign male Ags introduced at coitus. Seminal fluid profoundly influences cervical immune function, inducing proinflammatory cytokine synthesis and leukocyte recruitment. In this study, human Ect1 cervical epithelial cells and primary cervical cells were used to investigate agents in human seminal plasma that induce a proinflammatory response. TGF-β1, TGF-β2, and TGF-β3 are abundant in seminal plasma, and Affymetrix microarray revealed that TGF-β3 elicits changes in Ect1 cell expression of several proinflammatory cytokine and chemokine genes, replicating principal aspects of the Ect1 response to seminal plasma. The differentially expressed genes included several induced in the physiological response of the cervix to seminal fluid in vivo. Notably, all three TGF-β isoforms showed comparable ability to induce Ect1 cell expression of mRNA and protein for GM-CSF and IL-6, and TGF-β induced a similar IL-6 and GM-CSF response in primary cervical epithelial cells. TGF-β neutralizing Abs, receptor antagonists, and signaling inhibitors ablated seminal plasma induction of GM-CSF and IL-6, but did not alter IL-8, CCL2 (MCP-1), CCL20 (MIP-3α), or IL-1α production. Several other cytokines present in seminal plasma did not elicit Ect1 cell responses. These data identify all three TGF-β isoforms as key agents in seminal plasma that signal induction of proinflammatory cytokine synthesis in cervical cells. Our findings suggest that TGF-β in the male partner's seminal fluid may influence cervical immune function after coitus in women, and potentially be a determinant of fertility, as well as defense from infection.

Transforming growth factor (TGF)-βs are dimeric polypeptides that have vital roles in regulating cell growth and differentiation. They signal by assembling a receptor heterotetramer composed of two TβRI:TβRII heterodimers. To investigate whether the two heterodimers bind and signal autonomously, one of the TGF-β protomers was substituted to block receptor binding. The substituted dimer, TGF-β3 WD, bound the TβRII extracellular domain and recruited the TβRI with affinities indistinguishable from TGF-β3, but with one-half the stoichiometry. TGF-β3 WD was further shown to retain one-quarter to one-half the signalling activity of TGF-β3 in three established assays for TGF-β function. Single-molecule fluorescence imaging with GFP-tagged receptors demonstrated a measurable increase in the proportion of TβRI and TβRII dimers upon treatment with TGF-β3, but not with TGF-β3 WD. These results provide evidence that the two TβRI:TβRII heterodimers bind and signal in an autonomous manner. They further underscore how the TGF-βs diverged from the bone morphogenetic proteins, the ancestral ligands of the TGF-β superfamily that signal through a RI:RII:RII heterotrimer.

OBJECTIVE

Diabetic cardiomyopathy is characterized by the production of a disorganized fibrotic matrix in the absence of coronary atherosclerosis and hypertension. We examined whether adhesion of cardiac fibroblasts to glycated collagens mediates the differentiation of pro-fibrotic myofibroblasts, which may contribute to cardiac fibrosis.

RESULTS

By microarray, we found that methylglyoxal-treated collagen selectively enhanced α11 integrin expression in human cardiac fibroblasts, while levels of other collagen-binding integrins (α1, α2, and α10) were unchanged. Similar increases in α11 integrin mRNA and protein expression were observed in cardiac fibroblasts from streptozotocin (STZ)-treated Sprague-Dawley rats. In human cardiac fibroblasts plated on methyglyoxal-treated collagen and in cardiac fibroblasts from diabetic rats, transforming growth factor (TGF)-β2 but not TGF-β1 or TGF-β3 was increased compared with controls. Knock-down of α11 integrin and TGF-β receptors with small-interfering RNA blocked the increased expression of TGF-β2, α-smooth muscle actin (α-SMA), and α11 integrin that were induced in cells plated on methylglyoxal-treated collagen. Further, inhibition of Smad3 signalling blocked methylglyoxal-collagen up-regulation of α11 integrin and α-SMA expression. Rats with STZ-induced diabetes exhibited increased phosphorylation of Smad3 in cardiac tissues compared with control rats.

CONCLUSIONS

Interactions between α11 integrins and the Smad-dependent TGF-β2 signalling may contribute to the formation of pro-fibrotic myofibroblasts and the development of a fibrotic interstitium in diabetic cardiomyopathy.

Mammary tumorigenesis and epithelial-mesenchymal transition (EMT) programs cooperate in converting transforming growth factor-β (TGF-β) from a suppressor to a promoter of breast cancer metastasis. Although previous reports associated β1 and β3 integrins with TGF-β stimulation of EMT and metastasis, the functional interplay and plasticity exhibited by these adhesion molecules in shaping the oncogenic activities of TGF-β remain unknown. We demonstrate that inactivation of β1 integrin impairs TGF-β from stimulating the motility of normal and malignant mammary epithelial cells (MECs) and elicits robust compensatory expression of β3 integrin solely in malignant MECs, but not in their normal counterparts. Compensatory β3 integrin expression also 1) enhances the growth of malignant MECs in rigid and compliant three-dimensional organotypic cultures and 2) restores the induction of the EMT phenotypes by TGF-β. Of importance, compensatory expression of β3 integrin rescues the growth and pulmonary metastasis of β1 integrin-deficient 4T1 tumors in mice, a process that is prevented by genetic depletion or functional inactivation of β3 integrin. Collectively our findings demonstrate that inactivation of β1 integrin elicits metastatic progression via a β3 integrin-specific mechanism, indicating that dual β1 and β3 integrin targeting is necessary to alleviate metastatic disease in breast cancer patients.

BACKGROUND

Rotator cuff tendon heals by formation of an interposed zone of fibrovascular scar tissue. Recent studies demonstrate that transforming growth factor-beta 3 (TGF-β(3)) is associated with tissue regeneration and "scarless" healing, in contrast to scar-mediated healing that occurs with TGF-β(1).

OBJECTIVE

Delivery of TGF-β(3) in an injectable calcium-phosphate matrix to the healing tendon-bone interface after rotator cuff repair will result in increased attachment strength secondary to improved bone formation and collagen organization and reduced scar formation of the healing enthesis.

METHODS

Controlled laboratory study.

METHODS

Ninety-six male Sprague-Dawley rats underwent unilateral detachment of the supraspinatus tendon followed by acute repair using transosseous suture fixation. Animals were allocated into 1 of 3 groups: (1) repair alone (controls, n = 32), (2) repair augmented by application of an osteoconductive calcium-phosphate (Ca-P) matrix only (n = 32), or (3) repair augmented with Ca-P matrix + TGF-β(3) (2.75 µg) at the tendon-bone interface (n = 32). Animals were euthanized at either 2 weeks or 4 weeks postoperatively. Biomechanical testing of the supraspinatus tendon-bone complex was performed at 2 and 4 weeks (n = 8 per group). Microcomputed tomography was utilized to quantitate bone microstructure at the repair site. The healing tendon-bone interface was evaluated with histomorphometry and immunohistochemical localization of collagen types I (COLI) and III (COLIII). Statistical analysis was performed using 2-way analysis of variance with significance set at P < .05.

RESULTS

There was significantly greater load to failure of the Ca-P matrix + TGF-β(3) group compared with matrix alone or untreated controls at 4 weeks postoperatively (P = .04). At 2 weeks, microcomputed tomography revealed a larger volume of newly formed bone present at the healing enthesis in both experimental groups compared with the control group. By 4 weeks, this newly formed, woven bone had matured into calcified, lamellar bone. Histomorphometric analysis demonstrated significantly greater fibrocartilage and increased collagen organization at the healing tendon-bone insertion site in both experimental groups compared with the control group at 2 weeks (P = .04). Over time, TGF-β(3) delivery led to greater COLI expression compared with COLIII at the healing enthesis, indicating a more favorable COLI to COLIII ratio with administration of TGF-β(3).

CONCLUSIONS

Augmentation with an osteoconductive Ca-P matrix at the tendon-bone repair site is associated with new bone formation, increased fibrocartilage, and improved collagen organization at the healing tendon-bone interface in the early postoperative period after rotator cuff repair. The addition of TGF-β(3) significantly improved strength of the repair at 4 weeks postoperatively and resulted in a more favorable COLI/COLIII ratio.

CONCLUSIONS

The delivery of TGF-β(3) with an injectable Ca-P matrix at the supraspinatus tendon footprint has promise to improve healing after soft tissue repair.

Our objective was to assess the rejuvenation effect of extracellular matrix (ECM) deposited by human bone marrow stromal cells (hBMSCs) on hBMSC expansion and tissue-specific lineage differentiation potential. Passage 5 hBMSCs were expanded on ECM or conventional plastic flasks (Plastic) for one passage. Cell number was counted and immunophenotype profiles were assessed using flow cytometry. Selected integrins and proliferation-related pathway signals were assessed using Western blot. The expanded cells were evaluated for their chondrogenic potential in a pellet culture system with TGF-β3-containing chondrogenic medium using gross morphology, histology, immunostaining, biochemical analysis, real-time polymerase chain reaction, Western blot, and biomechanical testing. ECM-expanded hBMSCs were further evaluated for their osteogenic potential using Alizarin Red S staining and alkaline phosphatase activity assay and for their adipogenic potential using Oil Red O staining. ECM-expanded hBMSCs exhibited an enhanced proliferation capacity and an acquired robust chondrogenic potential compared to those grown on Plastic. ECM expansion decreased intracellular reactive oxygen species and increased stage-specific embryonic antigen-4 expression in hBMSCs. ECM expansion also upregulated integrins α2 and β5 and induced a sustained activation of Erk1/2 and cyclin D1. Interestingly, upregulation of TGF-β receptor II during cell expansion and chondrogenic induction might be responsible for an enhanced chondrogenic potential in ECM-expanded hBMSCs. We also found that ECM-expanded hBMSCs had an increased osteogenic potential and decreased adipogenic capacity. ECM deposited by hBMSCs may be a promising approach to expand BMSCs from elderly patients for the treatment of large-scale bone defects through endochondral bone formation.

The serine/threonine kinase, protein kinase C-theta (PKC-theta), plays a central role in the activation and differentiation of Th2 cells while being redundant in CD4+ and CD8+ antiviral responses. Recent evidence indicates that PKC-theta may however be required for some T cell-driven autoimmune responses. We have investigated the role of PKC-theta in the induction of autoimmune myocarditis induced by either Coxsackie B3 virus infection or immunization with alpha-myosin/CFA (experimental autoimmune myocarditis (EAM)). PKC-theta-deficient mice did not develop EAM as shown by impaired inflammatory cell infiltration into the heart, reduced CD4+ T cell IL-17 production, and the absence of a myosin-specific Ab response. Comparatively, PKC-theta was not essential for both early and late-phase Coxsackie virus-induced myocarditis. We sought to find alternate pathways of immune stimulation that might reconcile the differential requirements for PKC-theta in these two disease models. We found systemic administration of the TLR ligand CpG restored EAM in PKC-theta-deficient mice. CpG could act directly upon TLR9-expressing T cells to restore proliferation and up-regulation of Bcl-x(L), but exogenous IL-6 and TGF-beta was required for Th17 cell differentiation. Taken together, these results indicate that TLR-mediated activation of T cells can directly overcome the requirement for PKC-theta signaling and, combined with the dendritic cell-derived cytokine milieu, can promote the development of autoimmunity.

Recent successes in deriving human-induced pluripotent stem cells (hiPSCs) allow for the possibility of studying human neurons derived from patients with neurological diseases. Concomitant inhibition of the BMP and TGF-β1 branches of the TGF-β signaling pathways by the endogenous antagonist, Noggin, and the small molecule SB431542, respectively, induces efficient neuralization of hiPSCs, a method known as dual-SMAD inhibition. The use of small molecule inhibitors instead of their endogenous counterparts has several advantages including lower cost, consistent activity, and the maintenance of xeno-free culture conditions. We tested the efficacy of DMH1, a highly selective small molecule BMP-inhibitor for its potential to replace Noggin in the neuralization of hiPSCs. We compare Noggin and DMH1-induced neuralization of hiPSCs by measuring protein and mRNA levels of pluripotency and neural precursor markers over a period of seven days. The regulation of five of the six markers assessed was indistinguishable in the presence of concentrations of Noggin or DMH1 that have been shown to effectively inhibit BMP signaling in other systems. We observed that by varying the DMH1 or Noggin concentration, we could selectively modulate the number of SOX1 expressing cells, whereas PAX6, another neural precursor marker, remained the same. The level and timing of SOX1 expression have been shown to affect neural induction as well as neural lineage. Our observations, therefore, suggest that BMP-inhibitor concentrations need to be carefully monitored to ensure appropriate expression levels of all transcription factors necessary for the induction of a particular neuronal lineage. We further demonstrate that DMH1-induced neural progenitors can be differentiated into β3-tubulin expressing neurons, a subset of which also express tyrosine hydroxylase. Thus, the combined use of DMH1, a highly specific BMP-pathway inhibitor, and SB431542, a TGF-β1-pathway specific inhibitor, provides us with the tools to independently regulate these two pathways through the exclusive use of small molecule inhibitors.

Previous studies have shown that a field of genetically altered but histologically normal tissue extends 1 cm or more from the margins of human breast tumors. The extent, composition and biological significance of this field are only partially understood, but the molecular alterations in affected cells could provide mechanisms for limitless replicative capacity, genomic instability and a microenvironment that supports tumor initiation and progression. We demonstrate by microarray, qRT-PCR and immunohistochemistry a signature of differential gene expression that discriminates between patient-matched, tumor-adjacent histologically normal breast tissues located 1 cm and 5 cm from the margins of breast adenocarcinomas (TAHN-1 and TAHN-5, respectively). The signature includes genes involved in extracellular matrix remodeling, wound healing, fibrosis and epithelial to mesenchymal transition (EMT). Myofibroblasts, which are mediators of wound healing and fibrosis, and intra-lobular fibroblasts expressing MMP2, SPARC, TGF-β3, which are inducers of EMT, were both prevalent in TAHN-1 tissues, sparse in TAHN-5 tissues, and absent in normal tissues from reduction mammoplasty. Accordingly, EMT markers S100A4 and vimentin were elevated in both luminal and myoepithelial cells, and EMT markers α-smooth muscle actin and SNAIL were elevated in luminal epithelial cells of TAHN-1 tissues. These results identify cellular processes that are differentially activated between TAHN-1 and TAHN-5 breast tissues, implicate myofibroblasts as likely mediators of these processes, provide evidence that EMT is occurring in histologically normal tissues within the affected field and identify candidate biomarkers to investigate whether or how field cancerization contributes to the development of primary or recurrent breast tumors.

All transforming growth factor-β (TGF-β) ligands are synthesised as precursor molecules consisting of a signal peptide, an N-terminal prodomain and a C-terminal mature domain. During synthesis, prodomains interact non-covalently with mature domains, maintaining the molecules in a conformation competent for dimerisation. Dimeric precursors are cleaved by proprotein convertases, and TGF-β ligands are secreted from the cell non-covalently associated with their prodomains. Extracellularly, prodomains localise TGF-β ligands within the vicinity of their target cells via interactions with extracellular matrix proteins, including fibrillin and perlecan. For some family members (TGF-β1, TGF-β2, TGF-β3, myostatin, GDF-11 and BMP-10), prodomains bind with high enough affinity to suppress biological activity. The subsequent mechanism of activation of these latent TGF-β ligands varies according to cell type and context, but all activating mechanisms directly target prodomains. Thus, prodomains control many aspects of TGF-β superfamily biology, and alterations in prodomain function are often associated with disease.

Keratoconus (KC) affects 1:2000 people and is a disorder where cornea thins and assumes a conical shape. Advanced KC requires surgery to maintain vision. The role of oxidative stress in KC remains unclear. We aimed to identify oxidative stress levels between human corneal keratocytes (HCKs), fibroblasts (HCFs) and keratoconus cells (HKCs). Cells were cultured in 2D and 3D systems. Vitamin C (VitC) and TGF-β3 (T3) were used for 4 weeks to stimulate self-assembled extracellular matrix (ECM). No T3 used as controls. Samples were analyzed using qRT-PCR and metabolomics. qRT-PCR data showed low levels of collagen I and V, as well as keratocan for HKCs, indicating differentiation to a myofibroblast phenotype. Collagen type III, a marker for fibrosis, was up regulated in HKCs. We robustly detected more than 150 metabolites of the targeted 250 by LC-MS/MS per condition and among those metabolites several were related to oxidative stress. Lactate levels, lactate/malate and lactate/pyruvate ratios were elevated in HKCs, while arginine and glutathione/oxidized glutathione ratio were reduced. Similar patterns found in both 2D and 3D. Our data shows that fibroblasts exhibit enhanced oxidative stress compared to keratocytes. Furthermore the HKC cells exhibit the greatest level suggesting they may have a myofibroblast phenotype.

BACKGROUND

Currently, there is huge research focus on the development of novel cell-based regeneration and tissue-engineering therapies for the treatment of intervertebral disc degeneration and the associated back pain. Both bone marrow-derived (BM) mesenchymal stem cells (MSCs) and adipose-derived MSCs (AD-MSCs) are proposed as suitable cells for such therapies. However, currently no consensus exists as to the optimum growth factor needed to drive differentiation to a nucleus pulposus (NP)-like phenotype. The aim of this study was to investigate the effect of growth differentiation factor-6 (GDF6), compared with other transforming growth factor (TGF) superfamily members, on discogenic differentiation of MSCs, the matrix composition, and micromechanics of engineered NP tissue constructs.

METHODS

Patient-matched human AD-MSCs and BM-MSCs were seeded into type I collagen hydrogels and cultured in differentiating media supplemented with TGF-β3, GDF5, or GDF6. After 14 days, quantitative polymerase chain reaction analysis of chondrogenic and novel NP marker genes and sulfated glycosaminoglycan (sGAG) content of the construct and media components were measured. Additionally, construct micromechanics were analyzed by using scanning acoustic microscopy (SAM).

RESULTS

GDF6 stimulation of BM-MSCs and AD-MSCs resulted in a significant increase in expression of novel NP marker genes, a higher aggrecan-to-type II collagen gene expression ratio, and higher sGAG production compared with TGF-β or GDF5 stimulation. These effects were greater in AD-MSCs than in BM-MSCs. Furthermore, the acoustic-wave speed measured by using SAM, and therefore tissue stiffness, was lowest in GDF6-stiumlated AD-MSC constructs.

CONCLUSIONS

The data suggest that GDF6 stimulation of AD-MSCs induces differentiation to an NP-like phenotype and results in a more proteoglycan-rich matrix. Micromechanical analysis shows that the GDF6-treated AD-MSCs have a less-stiff matrix composition, suggesting that the growth factor is inducing a matrix that is more akin to the native NP-like tissue. Thus, this cell and growth-factor combination may be the ideal choice for cell-based intervertebral disc (IVD)-regeneration therapies.

BACKGROUND

Transforming growth factor beta (TGF-β) plays major roles in tumorigenesis by regulating cell growth, epithelial-to-mesenchymal transition (EMT), migration/invasion and metastasis. The epithelial markers E-cadherin, claudin-3 and claudin-4, commonly decreased in human adenocarcinomas are actually up regulated during ovarian carcinogenesis. In human ovarian cancer TGF-β1 may either suppress or promote tumor progression, but whether other TGF-β isoforms (TGF-β2 and TGF-β3) exert similar effects is not known.

METHODS

In this study we investigated the ability of the TGF-β isoforms (TGF-β1-3) to induce proliferation and migration by BrdU labeling, scratch wound and trans-filter migration assays in the human serous adenocarcinoma cell-line NIH-OVCAR3. Transepithelial resistance was measured and EMT observed by light-microscopy. Expression of adherens-, tight-junction and EMT-related transcription factors was analyzed by qRT-PCR and immunoblotting.

RESULTS

All TGF-β isoforms dose-dependently inhibited NIH-OVCAR3 cell growth, stimulated tumor cell migration with similar efficiency. The mesenchymal marker N-cadherin and claudin-1 expression was induced and occludin down regulated. However, migrating cells retained an epithelial shape and E-cadherin expression. The E-cadherin repressor SNAIL mRNA levels remained low independently of TGF-β1-3 treatment while ZEB1 expression was enhanced.

CONCLUSIONS

TGF-β1, TGF-β2 and TGF-β3 promote migration of NIH-OVCAR3 ovarian cancer cells independently of cell proliferation and without conversion to a complete EMT phenotype. Epithelial ovarian cancer commonly metastasis to the surrounding tissue or inside the peritoneum rather than invading blood vessels to set distance metastasis. Our result raises the question whether ovarian cancer primarily spread via collective migration than via single cell invasion.

BACKGROUND

Systemic sclerosis (SSc) is an autoimmune inflammatory disorder of unknown etiology characterized by fibrosis of the skin and internal organs. Ang II (angiotensin II), a vasoconstrictive peptide, is a well-known inducer of kidney, heart, and liver fibrosis. The goal of this study was to investigate the profibrotic potential of Ang II in the mouse skin.

METHODS

Ang II was administered by subcutaneous osmotic mini pumps to C57BL/6 male mice. Collagen-content measurements were performed with Gomori Trichrome staining and hydroxyproline assay. The mRNA expression level of collagens, TGF-β1, TGF-β2, TGF-β3, CTGF, αSMA, CD3, Emr1, CD45/B220, MCP1, and FSP1 were quantified with real-time polymerase chain reaction (PCR). Immunostaining was performed for markers of inflammation and fibrosis, including, phospho-Smad2, αSMA, CD3, Mac3, CD45/B220, and CD163B. Fibrocytes were identified by double staining with CD45/FSP1 and CD45/PH4. Endothelial cells undergoing endothelial-to-mesenchymal transition (EndoMT) were identified by double staining with VE-cadherin/FSP1.

RESULTS

Ang II-infused mice develop prominent dermal fibrosis in the area proximal to the pump, as shown by increased collagen and CTGF mRNA levels, increased hydroxyproline content, and more tightly packed collagen fibers. In addition, elevated mRNA levels of TGF-β2 and TGF-β3 along with increased expression of pSmad2 were observed in the skin of Ang II-treated mice. Dermal fibrosis was accompanied by an increased number of infiltrating fibrocytes, and an increased number of αSMA-positive cells, as well as CD163B⁺ macrophages in the upper dermis. This correlated with significantly increased mRNA levels of αSMA, Emr1, and MCP1. Infiltration of CD3-, CD45/B220-, and Mac3-positive cells was observed mainly in the hypodermis. Furthermore, an increased number of double-positive VE-cadherin/FSP1 cells were detected in the hypodermis only.

CONCLUSIONS

This work demonstrates that Ang II induces both inflammation and fibrosis in the skin via MCP1 upregulation and accumulation of activated fibroblasts. Additionally, our data suggest that populations of these fibroblasts originate from circulating blood cells. Ang II infusion via osmotic minipumps could serve as a useful mouse model of skin fibrosis to gain new insights into pathogenic mechanisms and to test new antifibrotic therapies.

Wound healing (WH) impairment is a well-documented phenomenon in clinical and experimental diabetes. Sex hormones, in addition to a number of signaling pathways including transforming growth factor-β1 (TGF-β1)/Smads and TNF-α/NF-κB in macrophages and fibroblasts, appear to play a cardinal role in determining the rate and nature of WH. We hypothesized that a defect in resolution of inflammation and an enhancement in TNF-α/NF-κB activity induced by estrogen deficiency contribute to the impairment of TGF-β signaling and delayed WH in diabetes models. Goto-Kakizaki (GK) rats and full thickness excisional wounds were used as models for type 2 diabetes (T2D) and WH, respectively. Parameters related to the various stages of WH were assessed using histomorphometry, western blotting, real-time PCR, immunofluorescence microscopy and ELISA-based assays. Retarded re-epithelialization, suppressed angiogenesis, delayed wound closure, reduced estrogen level and heightened states of oxidative stress were characteristic features of T2D wounds. These abnormalities were associated with a defect in resolution of inflammation, shifts in macrophage phenotypes, increased β3-integrin expression, impaired wound TGF-β1 signaling (↓p-Smad2/↑Smad7) and enhanced TNF-α/NFκB activity. Human/rat dermal fibroblasts of T2D, compared to corresponding control values, displayed resistance to TGF-β-mediated responses including cell migration, myofibroblast formation and p-Smad2 generation. A pegylated form of soluble TNF receptor-1 (PEG-sTNF-RI) or estrogen replacement therapy significantly improved re-epithelialization and wound contraction, enhanced TGFβ/Smad signaling, and polarized the differentiation of macrophages toward an M2 or "alternatively" activated phenotype, while limiting secondary inflammatory-mediated injury. Our data suggest that reduced estrogen levels and enhanced TNF-α/NF-κB activity delayed WH in T2D by attenuating TGFβ/Smad signaling and impairing the resolution of inflammation; most of these defects were ameliorated with estrogen and/or PEG-sTNF-RI therapy.

Biodegradable oligo(poly(ethylene glycol) fumarate) (OPF) composite hydrogels have been investigated for the delivery of growth factors (GFs) with the aid of gelatin microparticles (GMPs) and stem cell populations for osteochondral tissue regeneration. In this study, a bilayered OPF composite hydrogel that mimics the distinctive hierarchical structure of native osteochondral tissue was utilized to investigate the effect of transforming growth factor-β3 (TGF-β3) with varying release kinetics and/or insulin-like growth factor-1 (IGF-1) on osteochondral tissue regeneration in a rabbit full-thickness osteochondral defect model. The four groups investigated included (i) a blank control (no GFs), (ii) GMP-loaded IGF-1 alone, (iii) GMP-loaded IGF-1 and gel-loaded TGF-β3, and (iv) GMP-loaded IGF-1 and GMP-loaded TGF-β3 in OPF composite hydrogels. The results of an in vitro release study demonstrated that TGF-β3 release kinetics could be modulated by the GF incorporation method. At 12weeks post-implantation, the quality of tissue repair in both chondral and subchondral layers was analyzed based on quantitative histological scoring. All groups incorporating GFs resulted in a significant improvement in cartilage morphology compared to the control. Single delivery of IGF-1 showed higher scores in subchondral bone morphology as well as chondrocyte and glycosaminoglycan amount in adjacent cartilage tissue when compared to a dual delivery of IGF-1 and TGF-β3, independent of the TGF-β3 release kinetics. The results suggest that although the dual delivery of TGF-β3 and IGF-1 may not synergistically enhance the quality of engineered tissue, the delivery of IGF-1 alone from bilayered composite hydrogels positively affects osteochondral tissue repair and holds promise for osteochondral tissue engineering applications.

The transforming growth factor beta (TGF-β) family forms a group of three isoforms, TGF-β1, TGF-β2, and TGF-β3, with their structure formed by interrelated dimeric polypeptide chains. Pleiotropic and redundant functions of the TGF-β family concern control of numerous aspects and effects of cell functions, including proliferation, differentiation, and migration, in all tissues of the human body. Amongst many cytokines and growth factors, the TGF-β family is considered a group playing one of numerous key roles in control of physiological phenomena concerning maintenance of metabolic homeostasis in the bone tissue. By breaking the continuity of bone tissue, a spread-over-time and complex bone healing process is initiated, considered a recapitulation of embryonic intracartilaginous ossification. This process is a cascade of local and systemic phenomena spread over time, involving whole cell lineages and various cytokines and growth factors. Numerous in vivo and in vitro studies in various models analysing cytokines and growth factors' involvement have shown that TGF-β has a leading role in the fracture healing process. This paper sums up current knowledge on the basis of available literature concerning the role of the TGF-β family in the fracture healing process.

Recapitulating the microstructure of the native human corneal stromal tissue is believed to be a key feature in successfully engineering the corneal tissue. The stratified multilayered collagen fibril lamellae with orthogonal orientation determine the robust biomechanical properties of this tissue, and the uniform collagen fibril size and interfibrillar spacing are critical to its optical transparency. The objective of this investigation was to develop a highly organized collagen-fibril construct secreted by human corneal stromal stem cells (hCSSCs) to mimic the human corneal stromal tissue. In culture on a highly aligned fibrous substrate made from poly(ester urethane) urea, the fibroblast growth factor-2 (FGF-2, 10 ng/mL) and transforming growth factor-beta 3 (TGF-β3, 0.1 ng/mL) impacted the organization and abundance of the secreted collagen fibril matrix. hCSSCs differentiated into keratocytes with significant upregulation of the typical gene markers, including KERA, B3GnT7, and CHST6. FGF-2 treatment stimulated hCSSCs to secrete collagen fibrils strongly aligned in a single direction, whereas TGF-β3 induced collagenous layers with orthogonal fibril orientation. The combination of FGF-2 and TGF-β3 induced multilayered lamellae with orthogonally oriented collagen fibrils, in a pattern mimicking the human corneal stromal tissue. The constructs were 60-70 μm thick and had an increased content of cornea-specific extracellular matrix components, including keratan sulfate, lumican, and keratocan. The approach of combining substrate cues with growth factor augmentation offers a new means to engineer well-organized, collagen-based constructs with an appropriate nanoscale structure for corneal repair and regeneration.

The tumor microenvironment plays a significant role in colitis-associated cancer (CAC). Intestinal myofibroblasts (IMFs) are cells in the intestinal lamina propria secreting factors that are known to modulate carcinogenesis; however, the physiological role of IMFs and signaling pathways influencing CAC have remained unknown. Tumor progression locus 2 (Tpl2) is a MAPK that regulates inflammatory and oncogenic pathways. In this study we addressed the role of Tpl2 in CAC using complete and tissue-specific ablation of Tpl2 in mutant mice. Tpl2-deficient mice did not exhibit significant differences in inflammatory burdens following azoxymethane (AOM)/dextran sodium sulfate (DSS) administration compared with wild-type mice; however, the mutant mice developed significantly increased numbers and sizes of tumors, associated with enhanced epithelial proliferation and decreased apoptosis. Cell-specific ablation of Tpl2 in IMFs, but not in intestinal epithelial or myeloid cells, conferred a similar susceptibility to adenocarcinoma formation. Tpl2-deficient IMFs upregulated HGF production and became less sensitive to the negative regulation of HGF by TGF-β3. In vivo inhibition of HGF-mediated c-Met activation blocked early, enhanced colon dysplasia in Tpl2-deficient mice, indicating that Tpl2 normally suppresses the HGF/c-Met pathway. These findings establish a mesenchyme-specific role for Tpl2 in the regulation of HGF production and suppression of epithelial tumorigenesis.

Dilated cardiomyopathy (DCM) as a consequence of viral myocarditis is a worldwide cause of morbidity and death. The deposition of matrix proteins, such as collagen, in the course of ongoing viral myocarditis results in cardiac remodeling and finally in cardiac fibrosis, the hallmark of DCM. To identify mediators of virus-induced cardiac fibrosis, microarray analysis was conducted in a murine model of chronic coxsackievirus B3 (CVB3) myocarditis. By this attempt, we identified connective tissue growth factor (CTGF) as a novel factor highly expressed in infected hearts. Further investigations by quantitative reverse transcription polymerase chain reaction and Western blot analysis confirmed a strong induction of cardiac CTGF expression in the course of CVB3 myocarditis. By in situ hybridization and immunohistochemistry, basal CTGF messenger ribonucleic acid (mRNA) and protein expression were confined in noninfected control hearts mainly to endothelial cells, whereas in CVB3-infected hearts, also numerous fibroblasts were found to express CTGF. Regulation of CTGF is known to be basically mediated by transforming growth factor (TGF)-beta. In the course of CVB3 myocarditis, CTGF upregulation coincided with increased cardiac TGF-beta and procollagen type I mRNA expression, preceding the formation of fibrotic lesions. In in vitro experiments, we found that downregulation of CVB3 replication by means of small interfering RNAs (siRNAs) reverses the upregulation of CTGF mRNA expression. In contrast, downregulation of CTGF by siRNA molecules did not significantly reduce viral load, indicating that CTGF is not essential for CVB3 life cycle. The significantly enhanced transcript levels of TGF-beta, CTGF, and procollagen type I in cultivated CVB3-infected primary cardiac fibroblasts substantiate the role of fibroblasts as a relevant cell population in cardiac remodeling processes. We conclude that CTGF is a crucial molecule in the development of fibrosis in ongoing enteroviral myocarditis. Thus, downregulation of cardiac CTGF expression may open novel therapeutic approaches counteracting the development of cardiac fibrosis and subsequent heart muscle dysfunction.

BACKGROUND

For current tissue engineering or regenerative medicine strategies, chondrocyte (CC)- or mesenchymal stem cell (MSC)-seeded constructs are typically cultured in normoxic conditions (20% oxygen). However, within the knee joint capsule a lower oxygen tension exists.

OBJECTIVE

The objective of this study was to investigate how CCs and infrapatellar fad pad derived MSCs will respond to a low oxygen (5%) environment in 3D agarose culture. Our hypothesis was that culture in a low oxygen environment (5%) will enhance the functional properties of cartilaginous tissues engineered using both cell sources.

METHODS

Cell-encapsulated agarose hydrogel constructs (seeded with CCs or infrapatellar fat pad (IFP) derived MSCs) were prepared and cultured in a chemically defined serum-free medium in the presence (CCs and MSCs) or absence (CCs only) of transforming growth factor-beta3 (TGF-β3) in normoxic (20%) or low oxygen (5%) conditions for 42 days. Constructs were assessed at days 0, 21 and 42 in terms of mechanical properties, biochemical content and histologically.

RESULTS

Low oxygen tension (5%) was observed to promote extracellular matrix (ECM) production by CCs cultured in the absence of TGF-β3, but was inhibitory in the presence of TGF-β3. In contrast, a low oxygen tension enhanced chondrogenesis of IFP constructs in the presence of TGF-β3, leading to superior mechanical functionality compared to CCs cultured in identical conditions.

CONCLUSIONS

Extrapolating the results of this study to the in vivo setting, it would appear that joint fat pad derived MSCs may possess a superior potential to generate a functional repair tissue in low oxygen tensions. However, in the context of in vitro cartilage tissue engineering, CCs maintained in normoxic conditions in the presence of TGF-β3 generate the most mechanically functional tissue.

BACKGROUND

Transforming growth factor (TGF)-β signaling pathway, may act both as a tumor suppressor and as a tumor promoter in pancreatic cancer, depending on tumor stage and cellular context. TGF-β pathway has been under intensive investigation as a potential therapeutic target in the treatment of cancer. We hypothesized a correlation between TGF-βR2/SMAD4 expression in the tumor, plasma TGF-β1 ligand level, genetic variation in TGF-B pathway and prognosis of pancreatic cancer.

METHODS

We examined TGF-βR2 and SMAD4 protein expression in biopsy or surgical samples from 91 patients with pancreatic ductal adenocarcinoma (PDAC) using immunohistochemistry. Plasma level of TGF-β1 was measured in 644 patients with PDAC using ELISA. Twenty-eight single nucleotide polymorphisms (SNP) of the TGF-β1, TGF-β2, TGF-β3, TGF-βR1, TGF-βR2, and SMAD4 genes were determined in 1636 patients with PDAC using the Sequenom method. Correlation between protein expression in the tumor, plasma TGF-β1 level, and genotypes with overall survival (OS) was evaluated with Cox proportional regression models.

RESULTS

The expression level of TGF-βR2 and SMAD4 as an independent marker was not associated with OS. However, patients with both low nuclear staining of TGF-βR2 and high nuclear staining of SMAD4 may have better survival (P = 0.06). The mean and median level of TGF-β1 was 15.44 (SD: 10.99) and 12.61 (interquartile range: 8.31 to 19.04) ng/ml respectively. Patients with advanced disease and in the upper quartile range of TGF-β1 level had significantly reduced survival than those with low levels (P = 0.02). A significant association of SMAD4 SNP rs113545983 with overall survival was observed (P<0.0001).

CONCLUSIONS

Our data provides valuable baseline information regarding the TGF-β pathway in pancreatic cancer, which can be utilized in targeted therapy clinical trials. High TGF-β1 plasma level, SMAD4 SNP or TGF-βR2/SMAD4 tumor protein expression may suggest a dependence on this pathway in patients with advanced pancreatic cancer.

Tendon injuries are often associated with significant dysfunction and disability due to tendinous tissue's very limited self-repair capacity and propensity for scar formation. Dental-derived mesenchymal stem cells (MSCs) in combination with appropriate scaffold material present an alternative therapeutic option for tendon repair/regeneration that may be advantageous compared to other current treatment modalities. The MSC delivery vehicle is the principal determinant for successful implementation of MSC-mediated regenerative therapies. In the current study, a co-delivery system based on TGF-β3-loaded RGD-coupled alginate microspheres was developed for encapsulating periodontal ligament stem cells (PDLSCs) or gingival mesenchymal stem cells (GMSCs). The capacity of encapsulated dental MSCs to differentiate into tendon tissue was investigated in vitro and in vivo. Encapsulated dental-derived MSCs were transplanted subcutaneously into immunocompromised mice. Our results revealed that after 4 weeks of differentiation in vitro, PDLSCs and GMSCs as well as the positive control human bone marrow mesenchymal stem cells (hBMMSCs) exhibited high levels of mRNA expression for gene markers related to tendon regeneration (Scx, DCn, Tnmd, and Bgy) via qPCR measurement. In a corresponding in vivo animal model, ectopic neo-tendon regeneration was observed in subcutaneous transplanted MSC-alginate constructs, as confirmed by histological and immunohistochemical staining for protein markers specific for tendons. Interestingly, in our quantitative PCR and in vivo histomorphometric analyses, PDLSCs showed significantly greater capacity for tendon regeneration than GMSCs or hBMMSCs (P < 0.05). Altogether, these findings indicate that periodontal ligament and gingival tissues can be considered as suitable stem cell sources for tendon engineering. PDLSCs and GMSCs encapsulated in TGF-β3-loaded RGD-modified alginate microspheres are promising candidates for tendon regeneration.