transforming-growth-factor-beta and Joint-Diseases

Myhre syndrome (MS) is a developmental disorder characterized by typical facial dysmorphism, thickened skin, joint limitation and muscular pseudohypertrophy. Other features include brachydactyly, short stature, intellectual deficiency with behavioral problems and deafness. We identified SMAD4 as the gene responsible for MS. The identification of SMAD4 mutations in Laryngotracheal stenosis, Arthropathy, Prognathism and Short stature (LAPS) cases supports that LAPS and MS are a unique entity. The long-term follow up of patients shows that these conditions are progressive with life threatening complications. All mutations are de novo and changing in the majority of cases Ile500, located in the MH2 domain involved in transcriptional activation. We further showed an impairment of the transcriptional regulation via TGFβ target genes in patient fibroblasts. Finally, the absence of SMAD4 mutations in three MS cases may support genetic heterogeneity.

Topics: Adolescent; Adult; Child; Child, Preschool; Cryptorchidism; Disease Progression; Facies; Female; Fibroblasts; Follow-Up Studies; Genetic Heterogeneity; Genotype; Growth Disorders; Hand Deformities, Congenital; Humans; Hypertrophy; Intellectual Disability; Joint Diseases; Male; Mutation; Phenotype; Smad4 Protein; Transcriptional Activation; Transforming Growth Factor beta

The development of new biological approaches based on cell and gene therapies, in combination with tissue engineering, may create innovative ways to treat various tissues of the musculoskeletal system. It is vital for practicing orthopaedic surgeons to understand the terminology, fundamental concepts, and current research in this burgeoning field so that they may practice their discipline in its fullest form. Such techniques, coupled with advances in cell biology and polymer chemistry, are resulting in novel approaches to treating musculoskeletal disorders in which surgeons, who have traditionally used the tools of excision and reconstruction to treat patients, may now serve as surgical gardeners who create microenvironments that are conducive for tissue regeneration. Gene therapy and tissue engineering applications for bone healing, articular disorders, and skeletal muscle diseases and injuries are currently being explored. This review is intended to update readers on the principles and current advances in muscle-based gene therapy and tissue engineering for the musculoskeletal system.

Topics: Animals; Bone Diseases; Bone Morphogenetic Protein 2; Bone Morphogenetic Proteins; Cell Transplantation; Forecasting; Genetic Therapy; Genetic Vectors; Humans; Injections, Intra-Articular; Joint Diseases; Muscle, Skeletal; Muscular Diseases; Muscular Dystrophies; Recombinant Fusion Proteins; Recombinant Proteins; Stem Cell Transplantation; Stem Cells; Tissue Engineering; Transforming Growth Factor beta; Wound Healing

This study aimed to explore the role of TGF-β in tendon-to-bone healing after anterior cruciate ligament (ACL) reconstruction using bone marrow-derived mesenchymal stem cells (BMSCs) through the TGF-β/MAPK signaling pathway in a New Zealand white rabbit model.. A total of 72 healthy male New Zealand white rabbits were selected for these experiments. Flow cytometry and immunofluorescence were used to detect the expression of BMSC surface markers, and qRT-PCR was performed to detect TGF-β mRNA expression. The ACL reconstruction model was established with autografts. The rabbits were randomly divided into the following groups: inhibition of TGF-β (inhibition), over-expression of TGF-β (over-expression), empty vector and untreated (n = 18 per group). Hematoxylineosin (HE) staining, toluidine blue staining and Masson trichrome staining were conducted to observe any chondrocyte-like cell growth, and biomechanical tests were used to calculate the maximum load and rigidity. Three-dimensional CT imaging and Western blotting were applied to detect changes in bone tunnel size and bone density and the expression levels of TGF-β/MAPK signaling pathway-related proteins, respectively.. CD90 and CD44 were positively expressed, while CD11b was not detected. Compared with the empty vector and untreated groups, TGF-β mRNA expression was significantly decreased in the inhibition group but increased in the over-expression group; the latter group had a larger number of fibroblasts, a tighter tendon-bone interface, an increased number of chondrocyte-like cells and fibrochondrocytes, and more collagen fibers than the inhibition, empty vector and untreated groups. Compared with the empty vector and untreated groups, the maximum load and rigidity; the CT values of bone tunnel and bone tunnel margin; and the protein expression levels of TGF-β, p-ERK1/2, p-p38, p-JNK, c-jun and c-myc were significantly down-regulated in the inhibition group but up-regulated in the over-expression group.. Our study indicated that up-regulating TGF-β expression in BMSCs from New Zealand white rabbits could promote tendon-to-bone healing after ACL reconstruction by regulating the TGF-β/MAPK signaling pathway.

Topics: Animals; Anterior Cruciate Ligament Reconstruction; Bone Marrow Cells; Cells, Cultured; Disease Models, Animal; Hyaluronan Receptors; Joint Diseases; Knee Joint; Male; Mesenchymal Stem Cell Transplantation; Mesenchymal Stem Cells; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Rabbits; Signal Transduction; Tendons; Thy-1 Antigens; Transforming Growth Factor beta; Transplantation, Autologous; Up-Regulation; Wound Healing

Leri's pleonosteosis (LP) is an autosomal dominant rheumatic condition characterised by flexion contractures of the interphalangeal joints, limited motion of multiple joints, and short broad metacarpals, metatarsals and phalanges. Scleroderma-like skin thickening can be seen in some individuals with LP. We undertook a study to characterise the phenotype of LP and identify its genetic basis.. Whole-genome single-nucleotide polymorphism genotyping in two families with LP defined microduplications of chromosome 8q22.1 as the cause of this condition. Expression analysis of dermal fibroblasts from affected individuals showed overexpression of two genes, GDF6 and SDC2, within the duplicated region, leading to dysregulation of genes that encode proteins of the extracellular matrix and downstream players in the transforming growth factor (TGF)-β pathway. Western blot analysis revealed markedly decreased inhibitory SMAD6 levels in patients with LP. Furthermore, in a cohort of 330 systemic sclerosis cases, we show that the minor allele of a missense SDC2 variant, p.Ser71Thr, could confer protection against disease (p<1×10(-5)).. Our work identifies the genetic cause of LP in these two families, demonstrates the phenotypic range of the condition, implicates dysregulation of extracellular matrix homoeostasis genes in its pathogenesis, and highlights the link between TGF-β/SMAD signalling, growth/differentiation factor 6 and syndecan-2. We propose that LP is an additional member of the growing 'TGF-β-pathies' group of musculoskeletal disorders, which includes Myhre syndrome, acromicric dysplasia, geleophysic dysplasias, Weill-Marchesani syndromes and stiff skin syndrome. Identification of a systemic sclerosis-protective SDC2 variant lays the foundation for exploration of the role of syndecan-2 in systemic sclerosis in the future.

Topics: Adult; Aged; Child, Preschool; Chromosomes, Human, Pair 8; Extracellular Matrix; Facies; Female; Fibroblasts; Gene Duplication; Gene Expression Profiling; Growth Differentiation Factor 6; Hand Deformities, Congenital; Humans; Infant; Joint Diseases; Male; Middle Aged; Ossification, Heterotopic; Phenotype; Scleroderma, Systemic; Signal Transduction; Syndecan-2; Transforming Growth Factor beta; Young Adult

Myhre syndrome (MS, MIM 139210) is a connective tissue disorder that presents with short stature, short hands and feet, facial dysmorphic features, muscle hypertrophy, thickened skin, and deafness. Recurrent missense mutations in SMAD4 encoding for a transducer mediating transforming growth factor β (TGF-β) signaling are responsible for MS. We found that MS fibroblasts showed increased SMAD4 protein levels, impaired matrix deposition, and altered expression of genes encoding matrix metalloproteinases and related inhibitors. Increased TGF-β signaling and progression of aortic root dilation in Marfan syndrome can be prevented by the antihypertensive drug losartan, a TGF-β antagonists and angiotensin-II type 1 receptor blocker. Herein, we showed that losartan normalizes metalloproteinase and related inhibitor transcript levels and corrects the extracellular matrix deposition defect in fibroblasts from MS patients. The results of this study may pave the way toward therapeutic applications of losartan in MS.

Topics: Adolescent; Adult; Child; Cryptorchidism; Extracellular Matrix; Facies; Female; Fibroblasts; Growth Disorders; Hand Deformities, Congenital; Humans; Hypertrophy; Intellectual Disability; Joint Diseases; Losartan; Metalloendopeptidases; Microfibrils; Mutation; Phosphorylation; Signal Transduction; Smad2 Protein; Smad4 Protein; Transforming Growth Factor beta; Young Adult

Although alterations in biomarkers of cartilage turnover in synovial fluid (SF) have been demonstrated in horses with osteochondrosis (OC), there have been few investigations of such alterations in animals <1 year old. In this study tarsocrural SF samples from foals aged 18, 22 and 52 weeks of age were assessed for: (1) 'turnover' biomarkers of type II collagen (CPII and C2C) and proteoglycan (CS846 and glycosaminoglycans [GAG]); (2) matrix metalloproteinase (MMP) activity; (3) insulin-like growth factor (IGF)-1; (4) transforming growth factor (TGF)-β1; (5) prostaglandin (PG) E(2); and (6) leukotriene B(4). Using a linear mixed model, the concentration of biomarkers was compared between animals that developed or did not develop radiographic evidence of OC at 24 or 48 weeks of age. The CPII:C2C ratio tended to be higher in OC-affected joints compared to controls at all ages, and this difference was statistically significant at 22 weeks of age. The concentrations of CS846 and IGF-1, and the CS846:GAG ratio were reduced in OC-affected joints relative to controls at 18 weeks of age only. At 52 weeks of age, the PGE(2) concentration was lower in joints with OC. Overall, there appears to be a consistent anabolic shift in type II collagen turnover in juvenile joints affected by OC. Aberrant proteoglycan turnover is not a hallmark of the late repair of this lesion but reduced concentrations of IGF-1 in SF may be associated with early-stage lesions.

Topics: Age Factors; Animals; Biomarkers; Collagen Type II; Female; Glycosaminoglycans; Horse Diseases; Horses; Insulin-Like Growth Factor I; Joint Diseases; Leukotriene B4; Male; Matrix Metalloproteinases; Osteochondrosis; Prostaglandins; Proteoglycans; Radiography; Synovial Fluid; Tarsal Joints; Transforming Growth Factor beta

Transforming growth factor-beta1 and fibroblast growth factor-2 play very important roles in fibroblast proliferation and collagen expression. These processes lead to the formation of joint adhesions through the SMAD and MAPK pathways, in which ERK2 is supposed to be crucial. Based on these assumptions, lentivirus (LV)-mediated small interfering RNAs (siRNAs) targeting ERK2 were used to suppress the proliferation and collagen expression of rat joint adhesion tissue fibroblasts (RJATFs). Among four siRNAs examined, siRNA1 caused an 84% reduction in ERK2 expression (p<0.01) and was selected as the most efficient siRNA for use in this study. In subsequent experiments, significant downregulation of types I and III collagen were observed by quantitative RT-PCR and Western blot analyses. MTT assays and flow cytometry revealed marked inhibition of RJATF proliferation, but no apoptosis. In conclusion, LV-mediated ERK2 siRNAs may represent novel therapies or drug targets for preventing joint adhesion formation.

Topics: Animals; Apoptosis; Cell Line; Cell Proliferation; Collagen Type I; Collagen Type III; Fibroblast Growth Factor 2; Fibroblasts; Genetic Therapy; Humans; Joint Diseases; Lentivirus; Mitogen-Activated Protein Kinase 1; Rats; RNA, Small Interfering; Transfection; Transforming Growth Factor beta

To examine the ability of cartilage-like tissue, generated ectopically in a diffusion chamber using recombinant human bone morphogenetic protein 2 (rHuBMP-2), to repair cartilage defects in rats.. Muscle-derived mesenchymal cells were prepared by dissecting thigh muscles of 19-day postcoital rat embryos. Cells were propagated in vitro in monolayer culture for 10 days and packed within diffusion chambers (10(6)/chamber) together with type I collagen (CI) and 0, 1, or 10 microg rHuBMP-2, and implanted into abdominal subfascial pockets of adult rats. Tissue pellets were harvested from the diffusion chambers at 2 days to 6 weeks after implantation, and examined by histology, by reverse transcription-polymerase chain reaction (PCR) for aggrecan, CII, CIX, CX, and CXI, MyoD1, and core binding factor a1/runt-related gene 2, and by real-time PCR for CII. Tissue pellets generated in the chamber 5 weeks after implantation were transplanted into a full-thickness cartilage defect made in the patellar groove of the same strain of adult rat.. In the presence of 10 microg rHuBMP-2, muscle-derived mesenchymal cells expressed CII messenger RNA at 4 days after transplantation, and a mature cartilage mass was formed 5 weeks after transplantation in the diffusion chamber. Cartilage was not formed in the presence of 1 microg rHuBMP-2 or in the absence of rHuBMP-2. Defects receiving cartilage engineered with 10 microg rHuBMP-2 were repaired and restored to normal morphologic condition within 6 months after transplantation.. This method of tissue engineering for repair of articular defects may preclude the need to harvest cartilage tissue prior to mosaic arthroplasty or autologous chondrocyte implantation. Further studies in large animals will be necessary to validate this technique for application in clinical practice.

Topics: Animals; Bone Morphogenetic Protein 2; Bone Morphogenetic Proteins; Cartilage, Articular; Cell Differentiation; Chondrocytes; Diffusion Chambers, Culture; Embryo, Mammalian; Humans; Joint Diseases; Knee Joint; Muscle, Skeletal; Rats; Rats, Inbred F344; Recombinant Proteins; Thigh; Tissue Engineering; Transforming Growth Factor beta

The mucopolysaccharidoses (MPS) are inherited metabolic disorders resulting from the defective catabolism of glycosaminoglycans. In this report, we find that the stimulation of MPS connective tissue cells by the inflammatory cytokines causes enhanced secretion of several matrix-degrading metalloproteinases (MMPs). In addition, expression of tissue inhibitor of metalloproteinase-1 was elevated, consistent with the enhanced MMP activity. These findings were not restricted to one particular MPS disorder or species, and are consistent with previous observations in animal models with chemically induced arthritis. Bromodeoxyuridine incorporation studies also revealed that MPS chondrocytes proliferated up to 5-fold faster than normal chondrocytes, and released elevated levels of transforming growth factor-beta, presumably to counteract the marked chondrocyte apoptosis and matrix degradation associated with MMP expression. Despite this compensatory mechanism, studies of endochondral ossification revealed a reduction in chondro-differentiation in the growth plates. Thus, although MPS chondrocytes grew faster, most of the newly formed cells were immature and could not mineralize into bone. Our studies suggest that altered MMP expression, most likely stimulated by inflammatory cytokines and nitric oxide, is an important feature of the MPS disorders. These data also identify several proinflammatory cytokines, nitric oxide, and MMPs as novel therapeutic targets and/or biomarkers of MPS joint and bone disease. This information should aid in the evaluation of existing therapies for these disorders, such as enzyme replacement therapy and bone marrow transplantation, and may lead to the development of new therapeutic approaches.

Topics: Animals; Antimetabolites; Apoptosis; Biomarkers; Bone Diseases; Bromodeoxyuridine; Cats; Cell Proliferation; Cells, Cultured; Chondrocytes; Cytokines; Disease Models, Animal; Dogs; Immunoassay; Immunohistochemistry; Inflammation; Joint Diseases; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Mucopolysaccharidosis VI; Mucopolysaccharidosis VII; Nitric Oxide; Osteonectin; Rats; Reverse Transcriptase Polymerase Chain Reaction; Tibia; Time Factors; Tissue Inhibitor of Metalloproteinase-1; Transforming Growth Factor beta

To determine molecular changes in the expression of insulin-like growth factor-I (IGF-I) and transforming growth factor-beta1 (TGF-beta1) in horses with osteochondrosis, and to characterize expression of matrix aggrecan and collagen types I, II, and X in articular cartilage of affected joints.. Articular cartilage from affected stifle or shoulder joints of 11 horses with naturally acquired osteochondrosis and corresponding joints of 11 clinically normal horses.. Harvested specimens were snap frozen in liquid nitrogen, and total RNA was isolated. Specimens were fixed in 4% paraformaldehyde for histologic examinations. Expression of matrix molecules was assessed by analysis of northern blots and in situ hybridization, using equine-specific cDNA probes and riboprobes, respectively. Expression of IGF-I and TGF-beta1 was assessed by use of noncompetitive quantitative polymerase chain reaction, in situ hybridization, and immunohistochemical analysis.. Cartilage obtained from osteochondrosis lesions had significantly greater expression of IGF-I, compared with normal cartilage. Expression of TGF-beta1 and collagen type I were higher, but not significantly so, in affected tissues. Expression of aggrecan or collagen types II and X did not differ between affected and clinically normal cartilage.. Increased expression of growth factors and collagen type I was found in cartilage from osteochondrosis lesions. However, this probably reflects a healing response to injured tissue rather than a primary alteration. Therefore, methods aimed at altering concentrations of growth factors in cartilage of growing horses would be unlikely to alter the incidence or progress of the disease.

Topics: Aggrecans; Animals; Blotting, Northern; Cartilage, Articular; Collagen; Extracellular Matrix Proteins; Gene Expression Regulation, Developmental; Horse Diseases; Horses; Immunohistochemistry; In Situ Hybridization; Insulin-Like Growth Factor I; Joint Diseases; Lectins, C-Type; Osteochondritis; Proteoglycans; Reverse Transcriptase Polymerase Chain Reaction; RNA; Transforming Growth Factor beta; Transforming Growth Factor beta1

Binding between equine plasma alpha-2-macroglobulin (alpha 2M) and several cytokines known to participate in inflammatory reactions in other species was initially examined. Plasma was obtained from 5 horses with various abnormalities. Samples, both untreated and after reaction with methylamine, were incubated with exogenous, radiolabeled, porcine-derived transforming growth factor-beta-1 (125I-TGF-beta 1), recombinant human interleukin-1-beta (125I-IL-1 beta), and recombinant human tumor necrosis factor-alpha (125I-rhTNF-alpha). They were then subjected to nondenaturing polyacrylamide gel electrophoresis (PAGE). Binding of the native (slow) and activated (fast) forms of alpha 2M to each cytokine was subjectively evaluated with autoradiography. Equine alpha 2M bound 125I-TGF-beta 1. However, poor or no binding was observed between alpha 2M and either of 125I-rhTNF-alpha or 125I-IL-1 beta. Synovial fluid was then obtained from 6 normal horses, 6 horses with septic arthritis, and 6 horses with degenerative joint disease. Untreated and methylamine-reacted samples were quantitatively examined for binding with 125I-TGF-beta 1, using the autoradiographic techniques described above and densitometry. Native and activated alpha 2M were also quantified by densitometry of PAGE gels. Native alpha 2M was significantly elevated in septic arthritis (6.4% to 29.5% of total protein detected) and degenerative joint disease (2.8% to 12.3%), compared to normal joints (0.9% to 4.2%). Activated alpha 2M, however, was not detected in untreated synovial fluid samples. In all plasma and joint fluid samples, whether untreated or reacted with methylamine, 125I-TGF-beta 1 bound predominantly to alpha 2M, and preferentially to the activated form of alpha 2M. In synovial fluid, the amount of 125I-TGF-beta 1 binding was proportional to the quantity of alpha 2M present. These results indicate that: 1) equine alpha 2M binds TGF-beta 1; 2) the native form of alpha 2M is present in both equine plasma and synovial fluid, and 3) alpha 2M is a major binding protein for TGF-beta 1 in equine synovial fluid. Therefore, alpha 2M may play a role in regulating this mediator of inflammation in equine joints.

Topics: alpha-Macroglobulins; Animals; Binding, Competitive; Horse Diseases; Horses; Inflammation; Joint Diseases; Synovial Fluid; Transforming Growth Factor beta

Topics: Adult; Female; Humans; Interleukin-6; Joint Diseases; Male; Middle Aged; Spinal Diseases; Transforming Growth Factor beta

To investigate the effects of adenovirus vector-mediated gene transduction of E. coli beta-galactosidase (LacZ), transforming growth factor-beta 1 (TGF-beta 1), and heat shock protein 70 (HSP 70) on human chondrocyte-like cell line (HCS-2/8).. We examined expression of transduced genes and their expression periods by 5 bromo-4-chloroindolyl-beta-D-galactoside (X-gal) staining. Northern blotting, ELISA, and Western blotting. To assess the influence of TGF-beta 1 and HSP70 gene transduction, the expression of mRNA of type II collagen, proteoglycan core protein, matrix metalloproteinase 3 (MMP3) and tissue inhibitor of matrix metalloproteinase 1 were examined by Northern blotting.. Staining with X-gal indicated that the genes were transduced into a majority of the cells. Expression of the transduced genes in the cells was continued for at least 21 days. Transduction of TGF-beta 1 gene enhanced mRNA expression of type II collagen and proteoglycan core protein, but suppressed MMP3 mRNA expression in the cells. Expression of HSP70 was also high. Enhanced expression of HSP70 elevated mRNA expression of proteoglycan core protein.. These results indicate adenovirus vector is useful in chondrocyte gene therapy, and it could be an efficient mediator of TGF-beta 1 and HSP70 gene transduction.

Topics: Adenoviridae; Aggrecans; beta-Galactosidase; Blotting, Northern; Cartilage; Cell Count; Collagen; DNA Primers; Extracellular Matrix Proteins; Genetic Therapy; Genetic Vectors; Glycoproteins; HSP70 Heat-Shock Proteins; Humans; Joint Diseases; Lectins, C-Type; Matrix Metalloproteinase 3; Proteoglycans; RNA, Messenger; Tissue Inhibitor of Metalloproteinases; Transduction, Genetic; Transforming Growth Factor beta

Recombinant human cytokines were examined for their effects on plasminogen activator inhibitor-1 (PAI-1) production by human articular cartilage and chondrocyte monolayer cultures. Cartilage and chondrocytes were cultured with and without added cytokines and the conditioned media assayed for PAI-1 by a specific enzyme-linked immunosorbent assay, and mRNA levels determined by Northern blot analysis. Tumor necrosis factor alpha (TNF alpha) reduced, and transforming growth factor-beta (TGF-beta) and basic fibroblast growth factor (bFGF) increased, the levels of PAI-1 antigen and mRNA in the culture fluids and cell extracts, respectively. The effects of TNF alpha and TGF-beta on PAI-1 antigen levels were both time- and concentration-dependent; optimum doses being 10-100 pM TNF alpha and 0.4-0.8 nM TGF-beta, with each cytokine exerting its effect on PAI-1 antigen levels within 8 h of addition to culture. TNF alpha (and interleukin-1 alpha) also countered the effects of TGF-beta and bFGF. The anti-inflammatory drugs, indomethacin and dexamethasone, did not appear to modulate PAI-1 levels in cultures of cartilage tissue. The inhibition of PAI-1 levels by cytokines and reagents which stimulate cartilage resorption (i.e., TNF alpha, interleukin-1 alpha, retinoic acid) and enhancement by cytokines which counter it (i.e., TGF-beta, bFGF) further implicate plasminogen activator in the mechanism(s) of cartilage degradation in diseases such as arthritis.

Topics: Aged; Anti-Inflammatory Agents; Cartilage, Articular; Cells, Cultured; Cytokines; Dose-Response Relationship, Drug; Fibroblast Growth Factor 2; Humans; Joint Diseases; Plasminogen Activator Inhibitor 1; Plasminogen Activators; RNA, Messenger; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha; Up-Regulation