transforming-growth-factor-beta and Neurofibromatosis-1

Congenital tibial pseudarthrosis is a rare condition seen in neurofibromatosis type 1 (NF1), and treatment is complex. A randomized, placebo-controlled trial of bone morphogenetic protein (rhBMP-2; INFUSE bone graft) at time of tibial surgery was developed by the Neurofibromatosis Clinical Trials Consortium. Patients were randomized to receive rhBMP-2 that would, or would not, be added to the standard surgical procedure consisting of resection of pseudarthrosis tissue, insertion of a rigid intramedullary rod, and placement of autogenous iliac crest bone graft. Despite involvement of 16 centers with wide experience with NF1 orthopaedic management, only 5 patients (of 54 required) were able to be enrolled in the study during a 3-year time period. Because of the inability to recruit sufficient patients, this study was closed in June 2019, with plans to terminate. The obstacles that were encountered during the study are summarized. The authors question whether a randomized, placebo-controlled trial of a rare pediatric orthopaedic condition is possible to accomplish. Recommendations are provided to guide future studies of orthopaedic manifestations of NF1.Level of Evidence: Level V.

Topics: Bone Morphogenetic Protein 2; Bone Morphogenetic Proteins; Humans; Neurofibromatosis 1; Orthopedic Procedures; Patient Selection; Pseudarthrosis; Randomized Controlled Trials as Topic; Rare Diseases; Recombinant Proteins; Sample Size; Tibia; Transforming Growth Factor beta

Neurofibromas in von Recklinghausen's disease (vRD) can develop in the dermis. Therefore, we hypothesized that a dermal niche exists that promotes the development of these neurofibromas in subjects with vRD.. The purpose of this study is to examine the function of polydom, known as a ligand for integrin, mediating cell adhesion, and expressed in mouse nerve tissue, in promotion of neurofibroma.. Molecular, transcriptome and immunohistochemical analysis were performed to investigate the association between polydom expression and neurofibroma development.. Polydom mRNA levels were significantly higher in neurofibroma tissue than in control tissue. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis of RNA purified from primary cultured dermal neurofibroma cells demonstrated significantly higher polydom mRNA expression in cells derived from the surrounding dermis of neurofibromas compared to those from normal human dermal fibroblasts. RNA sequencing was used to compare gene expression between cultured cells derived from dermal neurofibroma-surrounding tissue with or without polydom knockdown. Subsequent gene ontology assays revealed that expression of integrinβ8 (ITGB8), a factor that releases transforming growth factor-β (TGF-β) from pro-TGF-β, was downregulated following polydom knockdown, suggesting upregulation of polydom-mediated TGF-β production. Furthermore, we observed a strong association between polydom expression and the increase in platelet-derived growth factor B (PDGFB) expression in primary cultured cells from the surrounding dermis of neurofibromas exposed to TGF-β1.. Our results suggest that increased polydom expression in the dermis surrounding neurofibromas may promote dermal neurofibroma development by activating the TGF-β signaling pathway.

Topics: Adolescent; Adult; Aged; Base Sequence; Cell Adhesion; Cell Adhesion Molecules; Dermis; Female; Fibroblasts; Gene Expression Regulation; Humans; Integrin beta Chains; Male; Middle Aged; Neurofibroma; Neurofibromatosis 1; Proto-Oncogene Proteins c-sis; RNA, Small Interfering; Signal Transduction; Transforming Growth Factor beta; Transforming Growth Factor beta1; Young Adult

Tibial pseudarthrosis associated with Neurofibromatosis type 1 (NF1) is an orthopedic condition with consistently poor clinical outcomes. Using a murine model that features localized double inactivation of the Nf1 gene in an experimental tibial fracture, we tested the effects of recombinant human bone morphogenetic protein-2 (rhBMP-2) and/or the bisphosphonate zoledronic acid (ZA). Tibiae were harvested at 3 weeks for analysis, at which time there was negligible healing in un-treated control fractures (7% union). In contrast, rhBMP-2 and rhBMP-2/ZA groups showed significantly greater union (87% and 93%, p < 0.01 for both). Treatment with rhBMP-2 led to a 12-fold increase in callus bone volume and this was further increased in the rhBMP-2/ZA group. Mechanical testing of the healed rhBMP-2 and rhBMP-2/ZA fractures showed that the latter group had significantly higher mechanical strength and was restored to that of the un-fractured contralateral leg. Co-treatment with rhBMP-2/ZA also reduced fibrous tissue infiltration at the fracture site compared to rhBMP alone (p = 0.068). These data support the future clinical investigation of this combination of anabolic and anti-resorptive agents for the treatment of NF1 pseudarthrosis. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:930-936, 2018.

Topics: Animals; Bone Density Conservation Agents; Bone Morphogenetic Protein 2; Bony Callus; Disease Models, Animal; Drug Evaluation, Preclinical; Drug Therapy, Combination; Female; Genes, Neurofibromatosis 1; Mice; Neurofibromatosis 1; Pseudarthrosis; Recombinant Proteins; Transforming Growth Factor beta; Zoledronic Acid

In congenital pseudarthrosis of the tibia, use of intramedullary (IM) fixation and autogenous bone graft has long been the standard of care. This study was undertaken to determine whether the addition of rhBMP-2 to this treatment method further enhances healing potential.. Twenty-one patients with congenital pseudarthrosis of the tibia were evaluated. Fifteen of these patients had neurofibromatosis type 1 (NF1). All had IM fixation and autogenous bone graft, followed by a BMP-soaked collagen sponge wrapped around both the fracture site and bone graft. A minimum 2 years' follow-up was required.. Follow-up averaged 7.2 years (range, 2.1 to 12.8 y). Sixteen of 21 tibias achieved bone union following the index surgery, at an average 6.6 months postoperatively. The 5 persistent nonunions occurred in NF1 patients. Further surgery was undertaken in these 5 NF1 patients, including the use of BMP. One of the 5 healed, 1 had persistent nonunion, and 3 eventually had amputation. Of the 16 patients who healed initially following the index surgery, 5 refractured (3 had NF1). Of these 5 patients, the IM fixation at the index surgery did not cross the ankle joint, and refracture occurred at the rod tip in 4. Three of these 5 patients healed following further surgery, 1 had persistent nonunion, and 1 had amputation. All of those with eventual amputation had NF1. No deleterious effects related to the use of BMP-2 were recognized in any patient.. The addition of rhBMP-2 appears to be helpful in shortening the time required to achieve fracture union in those who healed, but its use does not insure that healing will occur.. Level IV-therapeutic, case series.

Topics: Adolescent; Bone Morphogenetic Protein 2; Bone Transplantation; Child; Child, Preschool; Female; Fracture Fixation, Intramedullary; Humans; Infant; Male; Neurofibromatosis 1; Pseudarthrosis; Radiography; Recombinant Proteins; Retrospective Studies; Tibial Fractures; Transforming Growth Factor beta

Congenital tibial dysplasia is a severe pediatric condition that classically results in a persistent pseudarthrosis. A majority of these cases are associated with neurofibromatosis type I (NF1), a genetic disorder in which inactivation of the NF1 gene leads to overactivity of the Ras-MEK-MAPK (mitogen-activated protein kinase) signaling pathway. We therefore hypothesized that pharmaceutical inhibition of MEK-MAPK may be a beneficial therapeutic strategy.. Animals treated with the delivery vehicle alone, PD0325901, rhBMP-2, or the PD0325901 + rhBMP-2 combination showed union rates of 0%, 8%, 69% (p < 0.01), or 80% (p < 0.01), respectively, at twenty-one days after fracture. Mice treated with the rhBMP-2 + PD0325901 combination displayed a callus volume sixfold greater than the vehicle controls and twofold greater than the group receiving rhBMP-2 alone. Although MEK inhibition combined with rhBMP-2 led to increases in bone formation and union, the proportion of fibrous tissue in the callus was not significantly reduced.. The data suggest that MEK inhibition can promote bone formation in combination with rhBMP-2 in the context of an NF1 pseudarthrosis. However, PD0325901 did not promote substantive bone anabolism in the absence of an exogenous anabolic stimulus and did not suppress fibrosis.. This study examines a signaling pathway-based approach to treating poor bone healing in a model of NF1 pseudarthrosis.

Topics: Animals; Benzamides; Bone Morphogenetic Protein 2; Diphenylamine; Disease Models, Animal; Drug Therapy, Combination; Female; Mice; Mitogen-Activated Protein Kinase Kinases; Neurofibromatosis 1; Osteogenesis; Pseudarthrosis; Recombinant Proteins; Transforming Growth Factor beta

Neurofibromatosis type 1 (NF1) is a common genetic condition caused by mutations in the NF1 gene. Patients often suffer from tissue-specific lesions associated with local double-inactivation of NF1. In this study, we generated a novel fracture model to investigate the mechanism underlying congenital pseudarthrosis of the tibia (CPT) associated with NF1. We used a Cre-expressing adenovirus (AdCre) to inactivate Nf1 in vitro in cultured osteoprogenitors and osteoblasts, and in vivo in the fracture callus of Nf1(flox/flox) and Nf1(flox/-) mice. The effects of the presence of Nf1(null) cells were extensively examined. Cultured Nf1(null)-committed osteoprogenitors from neonatal calvaria failed to differentiate and express mature osteoblastic markers, even with recombinant bone morphogenetic protein-2 (rhBMP-2) treatment. Similarly, Nf1(null)-inducible osteoprogenitors obtained from Nf1 MyoDnull mouse muscle were also unresponsive to rhBMP-2. In both closed and open fracture models in Nf1(flox/flox) and Nf1(flox/-) mice, local AdCre injection significantly impaired bone healing, with fracture union being <50% that of wild type controls. No significant difference was seen between Nf1(flox/flox) and Nf1(flox/-) mice. Histological analyses showed invasion of the Nf1(null) fractures by fibrous and highly proliferative tissue. Mean amounts of fibrous tissue were increased upward of 10-fold in Nf1(null) fractures and bromodeoxyuridine (BrdU) staining in closed fractures showed increased numbers of proliferating cells. In Nf1(null) fractures, tartrate-resistant acid phosphatase-positive (TRAP+) cells were frequently observed within the fibrous tissue, not lining a bone surface. In summary, we report that local Nf1 deletion in a fracture callus is sufficient to impair bony union and recapitulate histological features of clinical CPT. Cell culture findings support the concept that Nf1 double inactivation impairs early osteoblastic differentiation. This model provides valuable insight into the pathobiology of the disease, and will be helpful for trialing therapeutic compounds.

Topics: Acid Phosphatase; Animals; Bone Morphogenetic Protein 2; Cell Differentiation; Cell Lineage; Cell Proliferation; Disease Models, Animal; Female; Fibrosis; Fracture Healing; Gene Deletion; HEK293 Cells; Humans; Integrases; Isoenzymes; Mice; Mice, Inbred C57BL; Mice, Knockout; Muscles; Neurofibromatosis 1; Neurofibromin 1; Osteoblasts; Osteoclasts; Osteogenesis; Pseudarthrosis; Recombinant Proteins; Reproducibility of Results; Tartrate-Resistant Acid Phosphatase; Tibia; Transforming Growth Factor beta

Neurofibromas are benign tumors that comprise primarily of Schwann cells and fibroblasts. Mast cells have been found scattered in the tumor tissue, and their role in promoting the proliferation of neurofibroma has been suggested. Tranilast (N-[3,4-dimethoxycinnamolyl]anthranilic acid) is an anti-allergic drug that inhibits release of the chemical mediators from mast cells and it used for the treatment of keloids and hypertrophic scars by its inhibition of growth-promoting transforming growth factor (TGF)-beta(1) from fibroblasts. We assumed that tranilast would suppress neurofibroma cell growth. In order to prove this hypothesis, we investigated the effectiveness of tranilast in inhibiting the tumor growth using a new cell culture system obtained from patients with neurofibromas. We called this culture system with the mixture of Schwann cells and fibroblasts "NF1 cells culture". Mast cells were differentiated from CD34(+) peripheral blood mononuclear cells of normal healthy subjects, and were co-cultured with NF1 cells. Three days after tranilast (10 approximately 100 microM) added to the culture dishes, we counted viable cell numbers and measured the concentrations of TGF-beta(1), stem cell factor (SCF) and tryptase, which exists in the histamine granule, in the culture medium. Tranilast significantly suppressed proliferation of the NF1 cells and lowered the levels of TGF-beta(1), SCF and tryptase. These results suggest that tranilast retards tumor proliferation through not only suppression of cell growth factor, but also the inhibition of a chemical mediator released from mast cells. Thus, tranilast can be a potent therapeutic agent to inhibit the growth of neurofibromas.

Topics: Anti-Allergic Agents; Cell Proliferation; Cells, Cultured; Down-Regulation; Humans; Mast Cells; Neurofibromatosis 1; ortho-Aminobenzoates; Stem Cell Factor; Transforming Growth Factor beta; Tryptases

Topics: Humans; Neurofibroma; Neurofibromatosis 1; Taurine; Transforming Growth Factor beta

Recombinant bone morphogenetic proteins (BMPs) show promise in treating the orthopedic complications associated with neurofibromatosis type 1 (NF1), such as congenital pseudarthrosis of the tibia. Minimal scientific information regarding the effects of BMP in the context of NF1 is available. As abnormalities in both bone formation and resorption have been documented in Nf1-deficient mice, we hypothesized that inadequate BMP-induced bone formation could be augmented by cotreatment with the bisphosphonate zoledronic acid (ZA). First, primary osteoblasts isolated from wild type (Nf1(+/+)) and Nf1-deficient (Nf1(+/-)) mice were cultured in the presence and absence of BMP-2. While Nf1(+/-) cells exhibited less osteogenic potential than Nf1(+/+) cells, alkaline phosphatase expression and matrix mineralization for both genotypes were enhanced by BMP-2 treatment. To model this response in vivo, 20 microg BMP-2 was implanted intramuscularly into the quadriceps of mice to induce heterotopic bone. Radiographs revealed significantly less net bone formation in Nf1(+/-) mice compared to Nf1(+/+) controls. To test the effect of an antiresorptive agent, mice were cotreated twice weekly from postoperative day 3 with 0.02 mg/kg ZA or with saline. ZA treatment led to a synergistic increase in the amount of heterotopic bone in both Nf1(+/+) and Nf1(+/-) mice compared with saline controls, as measured by DEXA and histomorphometry. Thus, the anabolic deficiency noted in Nf1(+/-) mice is amenable to stimulation by BMP-2, but mineralized tissue formation remains below that of Nf1(+/+) controls. Bisphosphonate combination therapy is superior to BMP therapy alone in terms of net bone production in vivo in both wild-type and Nf1-deficient mice.

Topics: Animals; Bone Density Conservation Agents; Bone Diseases, Metabolic; Bone Morphogenetic Protein 2; Bone Morphogenetic Proteins; Cell Survival; Cells, Cultured; Diphosphonates; Disease Models, Animal; Drug Therapy, Combination; Energy Metabolism; Female; Femur; Haplotypes; Imidazoles; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Neurofibromatosis 1; Neurofibromin 1; Osteoblasts; Skull; Tibia; Transforming Growth Factor beta; Zoledronic Acid

Neurofibromas are common tumors found in neurofibromatosis type 1 (NF1) patients. These complex tumors are composed of Schwann cells, mast cells, fibroblasts and perineurial cells embedded in collagen that provide a lattice for tumor invasion. Genetic studies demonstrate that in neurofibromas, nullizygous loss of Nf1 in Schwann cells and haploinsufficiency of Nf1 in non-neuronal cells are required for tumorigenesis. Fibroblasts are a major cellular constituent in neurofibromas and are a source of collagen that constitutes approximately 50% of the dry weight of the tumor. Here, we show that two of the prevalent heterozygous cells found in neurofibromas, mast cells and fibroblasts interact directly to contribute to tumor phenotype. Nf1+/- mast cells secrete elevated concentrations of the profibrotic transforming growth factor-beta (TGF-beta). In response to TGF-beta, both murine Nf1+/- fibroblasts and fibroblasts from human neurofibromas proliferate and synthesize excessive collagen, a hallmark of neurofibromas. We also establish that the TGF-beta response occurs via hyperactivation of a novel Ras-c-abl signaling pathway. Genetic or pharmacological inhibition of c-abl reverses fibroblast proliferation and collagen synthesis to wild-type levels. These studies identify a novel molecular target to inhibit neurofibroma formation.

Topics: Animals; Benzamides; Bone Marrow Cells; Cell Movement; Cell Proliferation; Collagen; Culture Media, Conditioned; Embryo, Mammalian; Fibroblasts; Fibrosis; Haplotypes; Heterozygote; Humans; Imatinib Mesylate; Mast Cells; Mice; Mice, Inbred C57BL; Mice, Mutant Strains; Neurofibroma; Neurofibromatosis 1; Phenotype; Piperazines; Proto-Oncogene Proteins c-abl; Proto-Oncogene Proteins p21(ras); Pyrimidines; Signal Transduction; Transforming Growth Factor beta

To compare the expression of immunohistochemical variables between benign and malignant components of malignant peripheral nerve sheath tumour (MPNST) arising within neurofibroma.. Eight cases of MPNST arising within a neurofibroma, associated with neurofibromatosis type 1 (NF1), were studied. The areas of MPNST and neurofibroma were compared immunohistochemically with regard to the expression of proliferative activity (MIB-1), growth factors, p53, bcl-2, neural cell adhesion molecule (N-CAM), and CD34.. The expression of transforming growth factor beta 1 (TGF-beta 1), TGF-beta receptor type II, hepatocyte growth factor alpha (HGF-alpha), c-met, p53, and N-CAM was higher in the areas of MPNST than in the neurofibromatous areas in four, five, five, eight, five, and three of the eight cases, respectively. CD34 expression was lower in the areas of MPNST than in the neurofibroma areas in three of the eight cases.. On the basis of these findings, TGF-beta 1, HGF-alpha, and p53 might be involved in the malignant transformation of neurofibroma to MPNST.

Topics: Adolescent; Adult; Animals; Antigens, CD34; Female; Growth Substances; Hepatocyte Growth Factor; Humans; Immunohistochemistry; Male; Mice; Middle Aged; Neoplasms, Multiple Primary; Nerve Sheath Neoplasms; Neural Cell Adhesion Molecules; Neurofibromatosis 1; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Proto-Oncogene Proteins c-met; Rabbits; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Transforming Growth Factor beta; Tumor Suppressor Protein p53

Platelet-derived growth factor BB (PDGF-BB) and transforming growth factor beta are known to enhance the growth of neurofibroma-derived cells from neurofibromatosis type 1 (NF-1) patients.. This study aims to elucidate whether these growth factors and their receptors are up-regulated in NF-1 patients.. Tissues and culture cells from neurofibroma in NF-1 patients, nontumor lesions in NF-1 patients, and the skin of normal controls were collected. To evaluate the expressions of growth factors and their receptors, reverse-transcriptase polymerase chain reaction and immunohistochemistry were performed.. PDGF-beta receptor subunit was expressed in the neurofibroma tissues from NF-1 patients, but not in the nontumorous tissues from NF-1 patients or skin from normal controls. As for the other factors, there were no significant differences among these tissues. Neurofibroma sections also stained positive for the PDGF-beta receptor subunit.. The interaction of PDGF and PDGF-beta receptor subunit may be important in the tumorigenesis of NF-1.

Topics: Becaplermin; Cells, Cultured; Gene Expression Regulation, Neoplastic; Growth Substances; Humans; Immunohistochemistry; Neurofibromatosis 1; Platelet-Derived Growth Factor; Proto-Oncogene Proteins c-sis; Receptor, Platelet-Derived Growth Factor alpha; Receptor, Platelet-Derived Growth Factor beta; Receptors, Growth Factor; Reverse Transcriptase Polymerase Chain Reaction; RNA, Neoplasm; Skin; Transforming Growth Factor beta; Tumor Cells, Cultured

In neurofibromatosis type-1 (NF-1), abnormal growth regulation may be related to the formation of multiple neurofibromas. We investigated the growth responses of neurofibroma-derived cells (NF cells) and control skin fibroblasts to various growth factors. The responses to platelet-derived growth factor (PDGF-BB) and transforming growth factor-beta 1 (TGF-beta 1) in NF cells were significantly greater than those in control fibroblasts. The increased response to PDGF-BB in NF cells was accompanied by an increased number of PDGF beta receptors, which was demonstrated by both 125I PDGF-BB binding assay and immunoblotting analysis. The increased response to TGF-beta 1 was assumed to be mediated through PDGF-like protein induction; TGF-beta 1-treated NF cells produced greater amounts of 36-kD PDGF-like protein than TGF-beta 1-treated control fibroblasts. These observations suggest that certain growth factors, e.g., PDGF-BB and TGF-beta, may play some role in the development of neurofibromas in NF-1.

Topics: Becaplermin; Cell Division; Cells, Cultured; Fibroblasts; Humans; Immunoblotting; Kinetics; Neurofibroma; Neurofibromatosis 1; Platelet-Derived Growth Factor; Proto-Oncogene Proteins c-sis; Receptors, Platelet-Derived Growth Factor; Recombinant Proteins; Skin; Skin Neoplasms; Thymidine; Transforming Growth Factor beta; Tumor Cells, Cultured