Different in vitro activation methods for latent transforming growth factors (TGF)–β : considerable exogenous factors to promote higher mesenchymal-origin cell proliferation in a bioprocessing platform

Regenerative medicine includes two efficient techniques, namely tissue-engineering and cell-based therapy in order to repair tissue damage efficiently. Most importantly, huge numbers of autologous cells are required to deal these practices. Nevertheless, primary cells, from autologous tissue, grow very slowly while culturing in vitro; moreover, they lose their natural characteristics over prolonged culturing period. Transforming growth factors-beta (TGF-β) is a ubiquitous protein found biologically in its latent form, which prevents it from eliciting a response until conversion to its active form. In active form, TGF-β acts as a proliferative agent in many cell lines of mesenchymal origin in vitro. This article reviews on some of the important activation methods-physiochemical, enzyme-mediated, non-specific protein interaction mediated, and drug-induced- of TGF-β, which may be established as exogenous factors to be used in culturing medium to obtain extensive proliferation of primary cells.

Isolation of transforming growth factor-beta 2 cDNA from a fish, Cyprinus carpio by RT-PCR

Transforming Growth Factors-beta (TGF-beta s) have been described in many vertebrate species of amphibians, aves and mammals. In this report we demonstrate the presence of TGF-beta 2 in pisces. TGF-beta 2 has been cloned from a fish, Cyrinus carpio, by RT-PCR using degenerate oligonucleotide primers. Sequence analysis of the amplified product and alignment of the deduced amino acid sequence with the human TGF-beta 2 amino acid sequence revealed 81% and 93% identity in the precursor and the mature regions, respectively. The northern blot analysis of fish heart RNA shows a major messenger RNA species of about 8.0 kb and two messages of very low abundance of about 5.0 kb and 4.0 kb. The identification of TGF-beta 2 isoform in Pisces and it's high degree of homology with the mammalian isoform suggests that among all TGF-beta isoforms, TGF-beta 2 is the most conserved during evolution. (C) 1997 Elsevier Science B.V.

Suppression of transforming growth factor beta signalling aborts caerulein induced pancreatitis and eliminates restricted stimulation at high caerulein concentrations

BACKGROUND: Transforming growth factors betas (TGF-betas) are implicated in pancreatic tissue repair but their role in acute pancreatitis is not known. To determine whether endogenous TGF-betas modulate the course of caerulein induced acute pancreatitis, caerulein was administered to wild-type (FVB-/-) and transgenic mice that are heterozygous (FVB+/-) for expression of a dominant negative type II TGF-beta receptor. METHODS: After 7 hourly supramaximal injections of caerulein, the pancreas was evaluated histologically and serum was assayed for amylase and lipase levels. Next, the effects of caerulein on amylase secretion were determined in mouse pancreatic acini, and cholecystokinin (CCK) receptor expression was assessed. RESULTS: The normal mouse pancreas was devoid of inflammatory cells whereas the pancreas from transgenic mice contained lymphocytic infiltrates. Caerulein injection in wild-type mice resulted in 6- and 36-fold increases in serum amylase and lipase levels, respectively, increased serum trypsinogen activation peptide (TAP) levels, gross oedema and a marked inflammatory response in the pancreas that consisted mainly of neutrophils and macrophages. By contrast, FVB+/- mice exhibited minimal alterations in response to caerulein with attenuated neutrophil-macrophage infiltrates. Moreover, acini from FVB+/- mice did not exhibit restricted stimulation at high caerulein concentrations, even though CCK receptor mRNA levels were not decreased. CONCLUSION: Our findings indicate that a functional TGF-beta signalling pathway may be required for caerulein to induce acute pancreatitis and for the CCK receptor to induce acinar cell damage at high ligand concentrations. Our results also support the concept that restricted stimulation at high caerulein concentrations contributes to the ability of caerulein to induce acute pancreatitis.

The recombinant proregion of transforming growth factor beta1 (latency-associated peptide) inhibits active transforming growth factor beta1 in transgenic mice.

All three isoforms of transforming growth factors beta (TGF-betal, TGF-beta2, and TGF-beta3) are secreted as latent complexes and activated extracellularly, leading to the release of the mature cytokines from their noncovalently associated proregions, also known as latency-associated peptides (LAPs). The LAP region of TGF-beta1 was expressed in a baculovirus expression system and purified to homogeneity. In vitro assays of growth inhibition and gene induction mediated by TGF-beta3 demonstrate that recombinant TGF-beta1 LAP is a potent inhibitor of the activities of TGF-betal, -beta2, and -beta3. Effective dosages of LAP for 50% neutralization of TGF-beta activities range from 4.7- to 80-fold molar excess depending on the TGF-beta isoform and activity examined. Using 125I-labeled LAP, we show that the intraperitoneal application route is effective for systemic administration of LAP. Comparison of concentrations of LAP in tissues shows a homogenous pattern in most organs with the exception of heart and muscle, in which levels of LAP are 4- to 8-fold lower. In transgenic mice with elevated hepatic levels of bioactive TGF-betal, treatment with recombinant LAP completely reverses suppression of the early proliferative response induced by TGF-beta1 in remnant livers after partial hepatectomy. The results suggest that recombinant LAP is a potent inhibitor of bioactive TGF-beta both in vitro and in vivo, after intraperitoneal administration. Recombinant LAP should be a useful tool for novel approaches to study and therapeutically modulate pathophysiological processes mediated by TGF-beta3.

Complex flexibility of the transforming growth factor beta superfamily.

The transforming growth factors beta (TGF-beta s) are important modulators of growth and differentiation. They are intermolecular disulfide-bonded homodimeric molecules. The monomer fold has a conserved cystine knot and lacks a hydrophobic core. The biological specificity of a given member of the family is believed to be determined by the conformational flexibility of the variable loop regions of the monomer. The monomer subunit assembly in the dimer is stabilized mainly by hydrophobic contacts and a few hydrogen bonds. Since these interactions are nondirectional, we examined subunit assemblies of TGF-beta by using conformational analysis. The different subunit assemblies in TGF-beta 2 dimer were characterized in terms of the intersubunit disulfide torsion. Our analyses show that the subunit assemblies fall into two states: the crystallographically observed gauche+conformation and the previously not reported gauche--conformation, both having almost identical interaction energies. Furthermore, there is significant flexibility in the subunit assembly within the gauche+ and the gauche- states of the disulfide bond. The monomer subunit assembly is independent of the variations about the loop regions. The variations in the loop regions, coupled with flexibility in the monomer assembly, lead to a complex flexibility in the dimer of the TGF-beta superfamily. For the TGF-beta superfamily, the cystine knot acts as a scaffold and complex flexibility provides for biological selectivity. Complex flexibility might provide an explanation for the diverse range of biological activities that these important molecules display.

Interactions between Growth Factors and Integrins: Latent Forms of Transforming Growth Factor-β Are Ligands for the Integrin αvβ1

The multipotential cytokine transforming growth factor-β (TGF-β) is secreted in a latent form. Latency results from the noncovalent association of TGF-β with its processed propeptide dimer, called the latency-associated peptide (LAP); the complex of the two proteins is termed the small latent complex. Disulfide bonding between LAP and latent TGF-β–binding protein (LTBP) produces the most common form of latent TGF-β, the large latent complex. The extracellular matrix (ECM) modulates the activity of TGF-β. LTBP and the LAP propeptides of TGF-β (isoforms 1 and 3), like many ECM proteins, contain the common integrin-binding sequence RGD. To increase our understanding of latent TGF-β function in the ECM, we determined whether latent TGF-β1 interacts with integrins. A549 cells adhered and spread on plastic coated with LAP, small latent complex, and large latent complex but not on LTBP-coated plastic. Adhesion was blocked by an RGD peptide, and cells were unable to attach to a mutant form of recombinant LAP lacking the RGD sequence. Adhesion was also blocked by mAbs to integrin subunits αv and β1. We purified LAP-binding integrins from extracts of A549 cells using LAP bound to Sepharose. αvβ1 eluted with EDTA. After purification in the presence of Mn2+, a small amount of αvβ5 was also detected. A549 cells migrated equally on fibronectin- and LAP-coated surfaces; migration on LAP was αvβ1 dependent. These results establish αvβ1 as a LAP-β1 receptor. Interactions between latent TGF-β and αvβ1 may localize latent TGF-β to the surface of specific cells and may allow the TGF-β1 gene product to initiate signals by both TGF-β receptor and integrin pathways.

Vitronectin modulates human mesenchymal stem cell response to insulin-like growth factor-I and transforming growth factor beta 1 in a serum-free environment

The use of animal sera for the culture of therapeutically important cells impedes the clinical use of the cells. We sought to characterize the functional response of human mesenchymal stem cells (hMSCs) to specific proteins known to exist in bone tissue with a view to eliminating the requirement of animal sera. Insulin-like growth factor-I (IGF-I), via IGF binding protein-3 or -5 (IGFBP-3 or -5) and transforming growth factor-beta 1 (TGF-beta(1)) are known to associate with the extracellular matrix (ECM) protein vitronectin (VN) and elicit functional responses in a range of cell types in vitro. We found that specific combinations of VN, IGFBP-3 or -5, and IGF-I or TGF-beta(1) could stimulate initial functional responses in hMSCs and that IGF-I or TGF-beta(1) induced hMSC aggregation, but VN concentration modulated this effect. We speculated that the aggregation effect may be due to endogenous protease activity, although we found that neither IGF-I nor TGF-beta(1) affected the functional expression of matrix metalloprotease-2 or -9, two common proteases expressed by hMSCs. In summary, combinations of the ECM and growth factors described herein may form the basis of defined cell culture media supplements, although the effect of endogenous protease expression on the function of such proteins requires investigation.

Transforming growth factor -beta signaling through Smad3 in allergy : Studies in the mechanisms of asthma, atopic dermatitis and allergic contact reactions

Transforming growth factor β signalling through Smad3 in allergy Allergic diseases, such as atopic dermatitis, asthma, and contact dermatitis are complex diseases influenced by both genetic and environmental factors. It is still unclear why allergy and subsequent allergic disease occur in some individuals but not in others. Transforming growth factor (TGF)-β is an important immunomodulatory and fibrogenic factor that regulates cellular processes in injured and inflamed skin. TGF-β has a significant role in the regulation of the allergen-induced immune response participating in the development of allergic and asthmatic inflammation. TGF-β is known to be an immunomodulatory factor in the progression of delayed type hypersensitivity reactions and allergic contact dermatitis. TGF-β is crucial in regulating the cellular responses involved in allergy, such as differentiation, proliferation and migration. TGF-β signals are delivered from the cytoplasm to the nucleus by TGF-β signal transducers called Smads. Smad3 is a major signal transducer in TGF-β -signalling that controls the expression of target genes in the nucleus in a cell-type specific manner. The role of TGF-β-Smad3 -signalling in the immunoregulation and pathophysiology of allergic disorders is still poorly understood. In this thesis, the role of TGF-β-Smad -signalling pathway using Smad3 -deficient knock out mice in the murine models of allergic diseases; atopic dermatitis, asthma and allergic contact reactions, was examined. Smad3-pathway regulates allergen induced skin inflammation and systemic IgE antibody production in a murine model atopic dermatitis. The defect in Smad3 -signalling decreased Th2 cytokine (IL-13 and IL-5) mRNA expression in the lung, modulated allergen induced specific IgG1 response, and affected mucus production in the lung in a murine model of asthma. TGF-β / Smad3 -signalling contributed to inflammatory hypersensitivity reactions and disease progression via modulation of chemokine and cytokine expression and inflammatory cell recruitment, cell proliferation and regulation of the specific antibody response in a murine model of contact hypersensitivity. TGF-β modulates inflammatory responses - at least partly through the Smad3 pathway - but also through other compensatory, non-Smad-dependent pathways. Understanding the effects of the TGF-β signalling pathway in the immune system and in disease models can help in elucidating the multilevel effects of TGF-β. Unravelling the mechanisms of Smad3 may open new possibilities for treating and preventing allergic responses, which may lead to severe illness and loss of work ability. In the future the Smad3 signalling pathway might be a potential target in the therapy of allergic diseases.

Chondrogenesis of adult stem cells from adipose tissue and bone marrow: induction by growth factors and cartilage-derived matrix.

OBJECTIVES: Adipose-derived stem cells (ASCs) and bone marrow-derived mesenchymal stem cells (MSCs) are multipotent adult stem cells with potential for use in cartilage tissue engineering. We hypothesized that these cells show distinct responses to different chondrogenic culture conditions and extracellular matrices, illustrating important differences between cell types. METHODS: Human ASCs and MSCs were chondrogenically differentiated in alginate beads or a novel scaffold of reconstituted native cartilage-derived matrix with a range of growth factors, including dexamethasone, transforming growth factor beta3, and bone morphogenetic protein 6. Constructs were analyzed for gene expression and matrix synthesis. RESULTS: Chondrogenic growth factors induced a chondrocytic phenotype in both ASCs and MSCs in alginate beads or cartilage-derived matrix. MSCs demonstrated enhanced type II collagen gene expression and matrix synthesis as well as a greater propensity for the hypertrophic chondrocyte phenotype. ASCs had higher upregulation of aggrecan gene expression in response to bone morphogenetic protein 6 (857-fold), while MSCs responded more favorably to transforming growth factor beta3 (573-fold increase). CONCLUSIONS: ASCs and MSCs are distinct cell types as illustrated by their unique responses to growth factor-based chondrogenic induction. This chondrogenic induction is affected by the composition of the scaffold and the presence of serum.

The influence of transforming growth factor-beta1 gene polymorphisms on the severity of gingival overgrowth associated with concomitant use of cyclosporin A and a calcium channel blocker

The purpose of this study was to determine whether the prevalence and severity of gingival overgrowth in renal transplant recipients concomitantly treated with cyclosporin and a calcium channel blocker was associated with functional polymorphisms within the signal sequence of the transforming growth factor-(TGF)beta1 gene.

Bone growth into a ceramic-filled defect around an implant - The response to transforming growth factor beta 1

Synthetic bone substitutes provide an alternative to autograft but do not give equivalent clinical results. Their performance may be enhanced by adding osteogenic growth factors. In this study, TGFbeta1 was absorbed on to a carrier of 0 tricalcium phosphate and Gelfoam(R) and used to fill a defect around a tibial implant in a rat model of revision arthoplasty.

Nuclear factor I-C links platelet-derived growth factor and transforming growth factor beta1 signaling to skin wound healing progression.

Transforming growth factor beta (TGF-beta) and platelet-derived growth factor A (PDGFAlpha) play a central role in tissue morphogenesis and repair, but their interplay remain poorly understood. The nuclear factor I C (NFI-C) transcription factor has been implicated in TGF-beta signaling, extracellular matrix deposition, and skin appendage pathologies, but a potential role in skin morphogenesis or healing had not been assessed. To evaluate this possibility, we performed a global gene expression analysis in NFI-C(-/-) and wild-type embryonic primary murine fibroblasts. This indicated that NFI-C acts mostly to repress gene expression in response to TGF-beta1. Misregulated genes were prominently overrepresented by regulators of connective tissue inflammation and repair. In vivo skin healing revealed a faster inflammatory stage and wound closure in NFI-C(-/-) mice. Expression of PDGFA and PDGF-receptor alpha were increased in wounds of NFI-C(-/-) mice, explaining the early recruitment of macrophages and fibroblasts. Differentiation of fibroblasts to contractile myofibroblasts was also elevated, providing a rationale for faster wound closure. Taken together with the role of TGF-beta in myofibroblast differentiation, our results imply a central role of NFI-C in the interplay of the two signaling pathways and in regulation of the progression of tissue regeneration.

Matrix metalloproteinases, TIMPs and growth factors regulating ameloblastoma behaviour

Aims: Ameloblastoma is an odontogenic neoplasm with local invasiveness and recurrence. We have previously suggested that growth factors and matrix metalloproteinases (MMPs) influence ameloblastoma invasiveness(1). The aim was to study expression of MMPs, tissue inhibitor of metalloproteinases (TIMPs) and growth factors in ameloblastoma. Methods and results: Thirteen cases of solid/multicystic ameloblastoma were examined. As a control, calcifying cystic odontogenic tumour (CCOT), a non-invasive odontogenic neoplasm with ameloblastomatous epithelium was also studied. Immunohistochemistry detected MMPs, TIMPs and growth factors in ameloblastoma and CCOT. The labelling index (LI) of MMP-9 and TIMP-2 was significantly higher in ameloblastoma compared with CCOT. The LI of epidermal growth factor (EGF), transforming growth factor (TGF)-alpha and epidermal growth factor receptor (EGFR) was also increased in ameloblastoma. This neoplasm showed greater expression of MMPs, TIMPs and growth factors compared with CCOT. We then analysed these molecules in ameloblastoma cells and stroma. Ameloblastoma cells exhibited increased LI of MMP-1, -2 and EGFR. We found a positive correlation between EGF and TIMP-1, and between TGF-alpha and TIMP-2. It is known that signals generated by growth factors are transduced by the ERK pathway. Ameloblastoma stroma exhibited the phosphorylated (activated) form of ERK. Conclusions: These results suggest an interplay involving growth factors MMPs and TIMPs that may contribute to ameloblastoma behaviour. Signals generated by this molecular network would be transduced by ERK 1/2 pathway.

Regulation of scleral cell contraction by transforming growth factor-β and stress competing roles in myopic growth

Reduced extracellular matrix accumulation in the sclera of myopic eyes leads to increased ocular extensibility and is related to reduced levels of scleral transforming growth factor-β (TGF-β). The current study investigated the impact of this extracellular environment on scleral cell phenotype and cellular biomechanical characteristics. Scleral cell phenotype was investigated in vivo in a mammalian model of myopia using the myofibroblast marker, α-smooth muscle actin (α-SMA). In eyes developing myopia α-SMA levels were increased, suggesting increased numbers of contractile myofibroblasts, and decreased in eyes recovering from myopia. To understand the factors regulating this change in scleral phenotype, the competing roles of TGF-β and mechanical stress were investigated in scleral cells cultured in three-dimensional collagen gels. All three mammalian isoforms of TGF-β altered scleral cell phenotype to produce highly contractile, α-SMA-expressing myofibroblasts (TGF-β3 > TGF-β2 > TGF-β1). Exposure of cells to the reduced levels of TGF-β found in the sclera in myopia produced decreased cell-mediated contraction and reduced α-SMA expression. These findings are contrary to the in vivo gene expression data. However, when cells were exposed to both the increased stress and the reduced levels of TGF-β found in myopia, increased α-SMA expression was observed, replicating in vivo findings. These results show that although reduced scleral TGF-β is a major contributor to the extracellular matrix remodeling in the myopic eye, it is the resulting increase in scleral stress that dominates the competing TGF-β effect, inducing increased α-SMA expression and, hence, producing a larger population of contractile cells in the myopic eye.

Healing and expression of growth factors (TGF-β and PDGF) in canine radial ostectomy gap containing platelet-rich plasma

Objectives: To evaluate bone healing in an experimental radial ostectomy in dogs treated with autologous platelet-rich plasma (PRP), through histological, densitometric, radiographic studies, as well as expression of growth factors in the ostectomy gap. Methods: Twenty-one dogs were randomly divided into either a control or a PRP group. All underwent unilateral ostectomy of the radius to generate a gap of 2.0 mm, that was stabilized with external skeletal fixation. The ostectomy gap was either filled with PRP or left empty as a control. The radiographic and densitometric studies were performed after surgery, then at intervals until 60 days during the post-surgery period. Histological and immunohistochemical evaluations were performed at seven or 60 days post-surgery. Analyses were performed using a statistical analysis system, and the level of significance was set at p <0.05. Results: The median radiographic healing score in the PRP group increased significantly between day 0 and day 60. Furthermore, at 60 days, the median healing score and the proportion of healed ostectomies in the control group (score 1; 1/6 healed) and the PRP group (score 5; 4/5 healed) were significantly different. There were differences between groups in radiographic and densitometric values at days 45 and 60. The histological evaluation showed advanced bone healing at 60 days in the PRP group and signs of delayed union in the control group. Clinical relevance: Platelet-rich plasma can be used as an adjuvant therapy because it may promote better bone healing of a radial ostectomy treated with external skeletal fixation in dogs.