Human Transforming Growth Factor-β1 Research Articles

Fibrin Scaffolds Perfused with Transforming Growth Factor-β1 as an In Vitro Model to Study Healthy and Tendinopathic Human Tendon Stem/Progenitor Cells.

A limited understanding of tendon cell biology in healthy and pathological conditions has impeded the development of effective treatments, necessitating in vitro biomimetic models for studying tendon events. We established a dynamic culture using fibrin scaffolds, bioengineered with tendon stem/progenitor cells (hTSPCs) from healthy or diseased human biopsies and perfused with 20 ng/mL of human transforming growth factor-β1 for 21 days. Both cell types showed long-term viability and upregulated Scleraxis (SCX-A) and Tenomodulin (TNMD) gene expressions, indicating tenogenic activity. However, diseased hTSPCs underexpressed collagen type I and III (COL1A1 and COL3A1) genes and exhibited lower SCX-A and TNMD protein levels, but increased type I collagen production, with a type I/type III collagen ratio > 1.5 by day 14, matching healthy cells. Diseased hTSPCs also showed constant high levels of pro-inflammatory cytokines, such as IL-8 and IL-6. This biomimetic environment is a valuable tool for studying tenogenic and inflammatory events in healthy and diseased tendon cells and identifying new therapeutic targets.

Sirt1 Inhibits Atrial Fibrosis by Downregulating the Expression of the Transforming Growth Factor-β1/Smad Pathway.

Atrial fibrosis is an important factor leading to atrial fibrillation, and the transforming growth factor-β1/Smad pathway is a key factor in inducing atrial fibrosis. Sirt1 is a member of the histone deacetylase (sirtuin) family, and recent studies have proven its cardioprotective effects. This study explored the effect of Sirt1 on atrial fibrosis through the transforming growth factor-β1/Smad pathway. We analyzed human right atrial appendage tissues and explored the relationship between Sirt1 and atrial fibrosis at the morphological, functional and molecular levels by Masson trichrome staining, immunofluorescence, real-time quantitative polymerase chain reaction and Western blot analysis. Rat atrial fibroblasts were extracted and treated by the Sirt1 agonist resveratrol, inhibitor sirtinol, and recombinant human transforming growth factor-β1 protein. The expression levels of related proteins were detected by Western blot, and the effect on the migration of atrial fibroblasts was detected by wound healing assay. We found that the expression of Sirt1 was reduced in the right atrial appendage tissues of patients with atrial fibrillation, and the degree of fibrosis was increased. In atrial fibroblasts, the activation of Sirt1 could inhibit the expression of transforming growth factor-β1/Smad and reduce the development of fibrosis, while inhibiting Sirt1 reduced its inhibitory effect on the transforming growth factor-β1/Smad pathway. These findings indicate that Sirt1 inhibits atrial fibrosis by downregulating the expression of the transforming growth factor-β1/Smad pathway, and provide potential targets for the treatment of atrial fibrillation.

Effect of Laurus nobilis on bacteria and human transforming growth factor-β1.

In this study, we aimed to determine the phenolic compounds, the antibacterial activity of extract from Laurus nobilis leaves, and its possible effect on transforming growth factor-β1 expression level in peripheral blood mononuclear cells. The phenolic components of Laurus nobilis were identified by the high-performance liquid chromatography method. The antibacterial activity of this extract was determined by disk diffusion and broth microdilution methods. The transforming growth factor-β1 expression was analyzed using the RT-qPCR method. Epicatechin was found in the highest amount and o-coumaric acid in the lowest amount. The half-maximal inhibitory concentration (IC50) was determined to be 55.17 μg/mL. The zones of inhibition and minimum inhibitory concentration for Staphylococcus aureus, Enterococcus faecalis, and Klebsiella pneumoniae were 15, 14, and 8 mm and 125, 250, and 1000 μg/mL, respectively. The change in transforming growth factor-β1 expression levels was found to be statistically significant compared with the control groups (p<0.0001). Laurus nobilis extract was found to be effective against bacteria and altered the expression level of transforming growth factor-β1 in peripheral blood mononuclear cells.

Osteochondral Repair with Autologous Cartilage Transplantation with or without Bone Grafting: A Short Pilot Study in Mini-Pigs.

Treatment strategies for osteochondral defects, for which particulated autologous cartilage transplantation (PACT) is an emerging treatment strategy, aim to restore the structure and function of the hyaline cartilage. Herein, we compared the efficacy of PACT with control or human transforming growth factor-β (rhTGF-β), and clarified the necessity of bone graft (BG) with PACT to treat shallow osteochondral defects in a porcine model. Two skeletally mature male micropigs received 4 osteochondral defects in each knee. The 16 defects were randomized to (1) empty control, (2) PACT, (3) PACT with BG, or (4) rhTGF-β. Animals were euthanized after 2 months and histomorphometry, immunofluorescence analysis, semiquantitative evaluation (O'Driscoll score), and magnetic resonance observation of cartilage repair tissue (MOCART) score were performed. Hyaline cartilages, glycosaminoglycan synthesis, and collagen type II staining were more abundant in the PACT than in the control and rhTGF-β groups. The O'Driscoll score was significantly different between groups (P < 0.001), with both PACT groups showing superiority (P = 0.002). PACT had the highest score (P = 0.002), with improved restoration of subchondral bone compared with PACT with BG. The MOCART score showed significant differences between groups (P = 0.021); MOCART and O'Driscoll scores showed high correlation (r = 0.847, P < 0.001). Treatment of osteochondral defects with PACT improved tissue quality compared with that with control or rhTGF-β in a porcine model. BG, in addition to PACT, may be unnecessary for shallow osteochondral defects. Clinical Relevance. BG may not be necessary while performing PACT.

Lymphatic Endothelial-to-Myofibroblast Transition: A Potential New Mechanism Underlying Skin Fibrosis in Systemic Sclerosis.

At present, only a few reports have addressed the possible contribution of the lymphatic vascular system to the pathogenesis of systemic sclerosis (SSc). Based on the evidence that blood vascular endothelial cells can undertake the endothelial-to-myofibroblast transition (EndMT) contributing to SSc-related skin fibrosis, we herein investigated whether the lymphatic endothelium might represent an additional source of profibrotic myofibroblasts through a lymphatic EndMT (Ly-EndMT) process. Skin sections from patients with SSc and healthy donors were immunostained for the lymphatic endothelial cell-specific marker lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1) in combination with α-smooth muscle actin (α-SMA) as the main marker of myofibroblasts. Commercial human adult dermal lymphatic microvascular endothelial cells (HdLy-MVECs) were challenged with recombinant human transforming growth factor-β1 (TGFβ1) or serum from SSc patients and healthy donors. The expression of lymphatic endothelial cell/myofibroblast markers was measured by quantitative real-time PCR, Western blotting and immunofluorescence. Collagen gel contraction assay was performed to assess myofibroblast-like cell contractile ability. Lymphatic endothelial cells in intermediate stages of the Ly-EndMT process (i.e., coexpressing LYVE-1 and α-SMA) were found exclusively in the fibrotic skin of SSc patients. The culturing of HdLy-MVECs with SSc serum or profibrotic TGFβ1 led to the acquisition of a myofibroblast-like morphofunctional phenotype, as well as the downregulation of lymphatic endothelial cell-specific markers and the parallel upregulation of myofibroblast markers. In SSc, the Ly-EndMT might represent a previously overlooked pathogenetic process bridging peripheral microlymphatic dysfunction and skin fibrosis development.

O-265 Increased CD8+ T-cell exhaustion due to high NKG2A/HLA-E engagement and IL-15 induction in adenomyosis

Abstract Study question Is there dysregulation of immune cells in the local uterine microenvironment of patients with adenomyosis? If any, what’s the underlying mediators and mechanisms? Summary answer Increased CD8+ T-cell exhaustion due to enhanced HLA-E/NKG2A engagement and IL-15 induction in ectopic adenomyotic lesions existed in human patients and mice models with adenomyosis. What is known already The immunological dysfunction has long been considered in the pathogenesis of adenomyosis. However, high heterogeneity in immune cell alterations and contradictory results were reported due to the technique limitation of immunohistochemistry mainly used in previous studies. A comprehensive picture of the dysregulation of immune cells in the local uterine microenvironment of patients with adenomyosis is still lacking. Specially, increased number of CD8+ T cells have been reported in adenomyotic patients, whether functional deficiency of CD8+ T cells may contribute to the abnormal accumulation and residency of ectopic endometrial cells at pathological sites remains unknown. Study design, size, duration The paired ectopic lesions and eutopic endometrium from 80 patients with adenomyosis, and normal endometrium and myometrium from 62 patients with uterine myoma (controls) were collected between 2021 to 2022. Mice models with adenomyosis by neonatal tamoxifen treatment were established for experiments in 2022. Participants/materials, setting, methods The alteration of immune subsets, NKG2A expression, exhausted phenotypes and CD8+ T-cell dysfunction by flow cytometry, the expression/localization of HLA-E and IL-15 by immunohistochemistry were investigated in uterine tissues of patients and mice models with adenomyosis. CD8+ T cells were sorted, and in-vitro cultured with recombinant human transforming growth factor-β (rhTGF-β) or rhIL-15 to determine the NKG2A expression. Correlation analyses were conducted for association between immune disturbances and disease severity (dysmenorrhea, local/diffuse lesion, CA125 level). Main results and the role of chance We found that an increase in CD8+ T cell number was the predominant alteration in ectopic lesions in patients with adenomyosis, and it was significantly associated with the severity of adenomyosis. For CD8+ T cells in adenomyotic lesions, the expression of NKG2A was positively correlated with CD94 expression, and CD94/NKG2A mainly enriched on CD103+ tissue resident CD8+ T subsets. The NKG2A+CD8+ T cells showed exhausted phenotypes characterized by co-expression of multiple immune inhibitory molecules (PD1, LAG3, TIM3, and TOX) and decreased degranulation capability demonstrated by reduced expression of perforin, granzyme B, and CD107. Importantly, the exhausted NKG2A+CD8+ T-cell subset was associated with disease severity and was significantly increased in ectopic uterine tissues of human patients and mice models with adenomyosis. The expression of NKG2A on CD8+ T cells was markedly upregulated by exogenous rhIL-15 but not rhTGF-β treatment. Additionally, we also found increased co-expression of IL-15 and the NKG2A ligand HLA-E on the glandular epithelial cells, where the NKG2A+CD8+ T cells were closely distributed around the gland, in ectopic adenomyotic microenvironment of patients and mice models with adenomyosis. Limitations, reasons for caution The control uterine samples were collected from patients with uterine myoma. Although they were pathologically confirmed as normal, the potential effects are unknown and cannot be excluded. Further studies in larger independent populations and exploration on the functional significance of CD8+ T-cell exhaustion in adenomyosis pathogenesis is warranted. Wider implications of the findings Our study reveals a previously unrecognized role for CD8+ T-cell exhaustion in the pathogenesis of adenomyosis and suggests that therapeutic interventions targeting and reinvigorating exhausted CD8+ T cells by NKG2A blockade may be beneficial for patients with adenomyosis. Trial registration number not applicable

Neoplastic tissue transfiguration in vivo by recombinant human transforming growth factor-β3

Human oral squamous cell carcinomas (hSCCs) are the most common head and neck cancers now presenting with more aggressive biological and clinical features due to smoking and alcohol together with widespread viremia. Transforming growth factor-β (TGF-β) proteins are powerful morphogens that induce rapid and substantial induction of endochondral bone formation but in primates only.

TGF-β3 Protects Neurons Against Intermittent Hypoxia-Induced Oxidative Stress and Apoptosis Through Activation of the Nrf-2/KEAP1/HO-1 Pathway via Binding to TGF-βRI.

Intermittent hypoxia (IH) is the primary pathological manifestation of obstructive sleep apnea (OSA) and the main cause of OSA-induced cognitive impairment. Hippocampal neurons are considered to be critical cells affected by IH. Transforming growth factor-β3 (TGF-β3) is a cytokine with a neuroprotective effect, which plays a crucial role in resisting hypoxic brain injury, while its role in IH-induced neuronal injury is still unclear. Here, we aimed to clarify the mechanism of TGF-β3 protecting IH-exposed neurons by regulating oxidative stress and secondary apoptosis. Morris water maze results revealed that IH exposure was unable to affect the vision and motor ability of rats, but significantly affected their spatial cognition. Second-generation sequencing (RNA-seq) and subsequent experiments supported that IH decreased TGF-β3 expression and stimulated reactive oxygen species (ROS)-induced oxidative stress and apoptosis in rat hippocampus. In vitro, IH exposure significantly activated oxidative stress within HT-22 cells. Exogenous administration of Recombinant Human Transforming Growth Factor-β3 (rhTGF-β3) prevented ROS surge and secondary apoptosis in HT-22 cells caused by IH, while TGF-β type receptor I (TGF-βRI) inhibitor SB431542 blocked the neuroprotective effect of rhTGF-β3. Nuclear factor erythroid 2-related factor 2 (Nrf-2) is a transcription factor preserving intracellular redox homeostasis. rhTGF-β3 improved the nuclear translocation of Nrf-2 and activated downstream pathway. However, Nrf-2 inhibitor ML385 suppressed the activation of the Nrf-2 mechanism by rhTGF-3 and restored the effects of oxidative stress damage. These results indicate that TGF-β3 binding to TGF-βRI activates the intracellular Nrf-2/KEAP1/HO-1 pathway, reduces ROS creation, and attenuates oxidative stress and apoptosis in IH-exposed HT-22 cells.

Transforming Growth Factorβ1 Overexpression Is Associated with Insulin Resistance and Rapidly Progressive Kidney Fibrosis under Diabetic Conditions.

Diabetes mellitus is a global health problem. Diabetic nephropathy is a common complication of diabetes mellitus and the leading cause of end-stage renal disease. The clinical course, response to therapy, and prognosis of nephropathy are worse in diabetic than in non-diabetic patients. The role of transforming growth factorβ1 in kidney fibrosis is undebatable. This study assessed whether the overexpression of transforming growth factorβ1 is associated with insulin resistance and the rapid progression of transforming growth factorβ1-mediated nephropathy under diabetic conditions. Diabetes mellitus was induced with streptozotocin in wild-type mice and transgenic mice with the kidney-specific overexpression of human transforming growth factorβ1. Mice treated with saline were the controls. Glucose tolerance and kidney fibrosis were evaluated. The blood glucose levels, the values of the homeostasis model assessment for insulin resistance, and the area of kidney fibrosis were significantly increased, and the renal function was significantly impaired in the diabetic transforming growth factorβ1 transgenic mice compared to the non-diabetic transgenic mice, diabetic wild-type mice, and non-diabetic mice. Transforming growth factorβ1 impaired the regulatory effect of insulin on glucose in the hepatocyte and skeletal muscle cell lines. This study shows that transforming growth factorβ1 overexpression is associated with insulin resistance and rapidly progressive kidney fibrosis under diabetic conditions in mice.

Pediatric Mandibular Defect Rehabilitation by Human Transforming Growth Factor-β3 with an Implant-Supported Prosthesis.

Large mandibular defects in children are an uncommon but challenging problem for surgeons to solve. The time-honored options of autologous bone grafts are seldom a viable option, as suitable donor sites are unavailable. Osteoinductive morphogens may yet provide a solution in these cases. A large mandibular tumor in a child 10 years of age necessitated the resection of the entire dentate portion of the mandible. The defect was reconstructed at a second stage with a composite graft of human transforming growth factor-β3 (hTGF-β3), human demineralized bone matrix, and 12 g of autologous bone harvested from the posterior iliac crest. A mature ossicle suitable for the placement of osseointegrated implants developed in the erstwhile defect, and an implant-supported dental prosthesis was placed. The patient has been followed up into adulthood. Facial growth has proceeded unhindered, and the patient has maintained full oral and dental function. This case reports the long-term result of an uncommon condition treated with a novel method. The longterm follow-up of this patient provides evidence to dispel some of the concerns for the use of osteoinductive proteins in children. A composite graft of osteogenic morphogens, osteocompetent autologous cells, and mineralized and demineralized matrices-as opposed to osteogenic morphogens used solo-may improve clinical bone regeneration.

Computational analysis of prodomain cysteines in human TGF-β proteins reveals frequent loss of disulfide-dependent regulation in tumors.

The functionally diverse members of the human Transforming Growth Factor-β (TGF-β) family are tightly regulated. TGF-β regulation includes 2 disulfide-dependent mechanisms-dimerization and partner protein binding. The specific cysteines participating in these regulatory mechanisms are known in just 3 of the 33 human TGF-β proteins. Human prodomain alignments revealed that 24 TGF-β prodomains contain conserved cysteines in 2 highly exposed locations. There are 3 in the region of the β8 helix that mediates dimerization near the prodomain carboxy terminus. There are 2 in the Association region that mediates partner protein binding near the prodomain amino terminus. The alignments predict the specific cysteines contributing to disulfide-dependent regulation of 72% of human TGF-β proteins. Database mining then identified 9 conserved prodomain cysteine mutations and their disease phenotypes in 7 TGF-β proteins. Three common adenoma phenotypes for prodomain cysteine mutations suggested 7 new regulatory heterodimer pairs. Two common adenoma phenotypes for prodomain and binding partner cysteine mutations revealed 17 new regulatory interactions. Overall, the analysis of human TGF-β prodomains suggests a significantly expanded scope of disulfide-dependent regulation by heterodimerization and partner protein binding; regulation that is often lost in tumors.

Abstract 1299: Immunotherapeutic hcw9218 enhances anti-tumor activity of chemotherapy by facilitating immune-mediated elimination of therapy induced senescent cells

Abstract Advances in immunostimulatory and anti-immunosuppressive therapeutics have revolutionized cancer treatment. Here, we report a novel heterodimeric bifunctional fusion molecule, HCW9218, constructed using our soluble tissue factor (TF)-based scaffold technology comprising extracellular domains of the human transforming growth factor-β (TGF-β) receptor II and a human interleukin (IL)-15/IL-15 receptor α complex. This fusion complex exhibited TGF-β neutralizing activity in vitro and sequestered plasma TGF-β in vivo. Subcutaneous administration of HCW9218 was well tolerated in mice, with a half-life sufficient to provide long lasting biological activity. HCW9218 enhanced metabolic and cytotoxic activities of immune cells (CD8+ T cells, NK cells) and reduced tumor progression in models of chemotherapy induced senescence (TIS, docetaxel for B16F10 tumors and gemcitabine plus nab-paclitaxel for SW1990 tumor models) and non-TIS in vivo. Mechanistically, HCW9218 treatment not only affected the immunosuppressive tumor microenvironment but also enhanced CD8+ T cells and NK cells infiltration and cytotoxicity in the tumors to eliminate TIS cancer cells. Immune-depletion studies showed that HCW9218-activated NK cells played a pivotal role in TIS cancer cell removal. HCW9218 treatment following docetaxel chemotherapy further enhanced efficacy of tumor antigen-specific and anti-PDL-1 antibodies in B16F10 tumor-bearing mice. We also show that HCW9218 treatment lowered senescence associated secretory phenotype (SASP) factors in off-target tissues in chemotherapy treated tumor-bearing mice. Thus, HCW9218 may serve as a novel therapeutic (monotherapy or in combination with chemotherapy) to simultaneously provide immunostimulation and lessen immunosuppression associated with tumors. HCW9218 has been cleared by the U.S. Food and Drug Administration to proceed for evaluation in a first-inhuman Phase 1b clinical trial in patients with advanced pancreatic cancer. Citation Format: Pallavi Chaturvedi, Varghese George, Bai Liu, Niraj Shrestha, Meng Wang, Micheal Dee, Xiaoyun Zhu, Jack Egan, Jin-an Jiao, Catherine Spanoudis, Jilan Xing, Victor Gallo, Christian Echeverri, Lijing You, Lin Kong, Gabriela Muniz, Peter Rhode, Hing Wong. Immunotherapeutic hcw9218 enhances anti-tumor activity of chemotherapy by facilitating immune-mediated elimination of therapy induced senescent cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1299.

Flavonoids from Stems and Leaves of Scutellaria baicalensis Georgi Regulate the Brain Tau Hyperphosphorylation at Multiple Sites Induced by Composited Aβ in Rats.

Neurofibrillary Tangles (NFTs), formed by hyperphosphorylation of Tau protein in Alzheimer's Disease (AD), arethe main pathomechanisms of neuronal degeneration, which indicate a sign of brain disorder. NFTs are positively correlated with the degree of cognitive impairment in AD. The objective of this study isto investigate the effect of flavonoids from the stems and leaves of Scutellaria baicalensis Georgi (SSF) on the hyperphosphorylated expression levels at multiple sites of Tau protein induced by β-amyloid protein 25-35 (Aβ25-35) in combination with aluminum trichloride (AlCl3) and recombinant human transforming growth factor-β1(RHTGF-β1) (composited Aβ) in rats. The AD rat models were established by intracerebroventricular injection of Aβ25-35 and AlCl3 combined with RHTGF-β1. On day 45, after the operation, the Morris water maze test was conducted to screen the memory impairment of AD models. The successful model rats were randomly divided into the model group and the three-dose drug group. The drug group rats were orally administered SSF daily for 38 days. Western blotting was performed to detect the protein expression of P-Tau (Thr 181), P-Tau (Thr 217), P-Tau (Thr 231), P-Tau (Ser 199), P-Tau (Ser 235), P-- Tau (Ser 396), and P-Tau (Ser 404) in the hippocampus and cerebral cortex of rats. Compared with the sham group, the expression of P-Tau (Thr 181), P-Tau (Thr 217), P-- Tau (Thr 231), P-Tau (Ser 199), P-Tau (Ser 235), P-Tau (Ser 396), and P-Tau (Ser 404)was significantly increased in the hippocampus and cerebral cortex of the model group (P < 0.01). However, the three doses of SSF, i.e., 35, 70, and 140 mg/kg, regulated the expression of phosphorylated Tau proteinto varying degrees in the hippocampus and cerebral cortex of AD model rats (P < 0.01). SSF could significantly reduce the protein expression levels of P-Tau (Thr 181), PTau (Thr 217), P-Tau (Thr 231), P-Tau (Ser 199), P-Tau (Ser 235), P-Tau (Ser 396), and P-Tau (Ser 404), induced by the intracerebroventricular injection of composited Aβ, in rats' brain. These results indicated that the neuro-protection and the improvement in the impaired memory of rats by SSF were due to the inhibition of hyperphosphorylation of Tau protein at multiple sites in rats' brain.

The Induction of Bone Formation by the recombinant human transforming growth Factor-β3 : From preclinical studies in Papio ursinus to translational research in Homo sapiens

Skeletal bone defects of the axial or the craniomaxillofacial skeletons still present formidable challenges to skeletal reconstructionists, tissue biologists and modern medicine. In systematic research experiments in the Chacma baboon Papio ursinus our laboratories have shown the previously unreported osteoinductive activity of the three mammalian transforming growth factor-β (TGF-β) isoforms. This review discusses the induction of bone formation by the mammalian TGF-βs with particular reference to the substantial and rapid induction of bone by the recombinant hTGF-β3 from the laboratory benches, to pre-clinical studies in heterotopic and orthotopic mandibular sites of Papio ursinus to clinical translation in human patients. Design and MethodsA series of systematic research experiments in Papio ursinus using the hTGF-β3 together with earlier experiments using the -β1 and β2 isoforms are reviewed and re-analyzed molecularly nd morphologically to provide the basic research data for the reported clinical translation in human patients. Results The three mammalian hTGF-β isoforms and notably hTGF-β3 induce rapid and substantial induction of heterotopic bone in intramuscular sites of Papio ursinus. Relatively low doses of hTGF-β1or hTGF-β3 in binary application with hBMP-7 synergize to induce massive corticalized ossicles in the rectus abdominis muscle. In orthotopic mandibular sites, 125 and250 µg doses of hTGF-β3 induce bone formation across large mandibular defects in Papio ursinus with corticalized buccal and lingual plates by day 30, with modeling and maintenance of corticalized bone by 9 to 12 months after implantation of the 250 µg dose in 3 cm mandibular defects Papio ursinus. Discussion hTGF-β3 significantly up-regulates RUNX-2 and Osteocalcin expression on day 15 controlling the differentiation of progenitor stem cells into the osteoblastic lineage. The induction of bone by the hTGF-β3 is via the bone morphogenetic proteins pathway; hTGF-β3 controls theinduction of bone by regulating the expression of BMPs gene and gene products via Noggin expression, eliciting bone induction by up-regulating exogenous BMPs.

Osteogenic Competence and Potency of the Bone Induction Principle: Inductive Substrates That Initiate "Bone: Formation by Autoinduction".

The de novo induction of bone has always been a fascinating phenomenon, keeping skeletal reconstructionists and cellular developmental biologists continuously engaged to finally provide a molecular and cellular approach to the induction of bone formation. A significant advancement was made by the purification and cloning of the human recombinant bone morphogenetic proteins, members of the transforming growth factor-β supergene family. Human bone morphogenetic proteins are powerful inducers of bone in animal models including nonhuman primates. Translation in clinical contexts has however, proven to be surprisingly difficult. This review also describes the significant induction of bone formation by the human transforming growth factor-β3 when implanted in heterotopic intramuscular sites of the Chacma baboon Papio ursinus. Large mandibular defects implanted with 250 mg human transforming growth factor-β3 in human patients showed significant osteoinduction; however, the induction of bone was comparatively less than the induction of bone in P ursinus once again highlighting the conundrum of human osteoinduction: is the bone induction principle failing clinical translation?

Mesenchymal Stem Cells With Cancer-Associated Fibroblast-Like Phenotype Stimulate SDF-1/CXCR4 Axis to Enhance the Growth and Invasion of B-Cell Acute Lymphoblastic Leukemia Cells Through Cell-to-Cell Communication.

Background: Bone marrow mesenchymal stem cells (BM-MSCs) are the stromal cells in the leukemia microenvironment, and can obtain cancer-associated fibroblast (CAF)-like phenotype under certain conditions to further promote leukemia progression. However, the mechanism of MSCs with CAF-like phenotype interacting with leukemia cells in B-cell acute lymphoblastic leukemia (B-ALL) and promoting the progression of B-ALL remains unclear.Methods: Mesenchymal stem cells with CAF-like phenotype were obtained by treating MSCs with recombinant human transforming growth factor-β (rhTGF-β), hereafter referred to as TGF-β conditioned MSCs. In vivo mouse model experiments, in vitro transwell chamber experiments, three-dimensional (3D) cell culture models, lentiviral transfection and other experimental methods were used to investigate the possible mechanism of the interaction between TGF-β conditioned MSCs and leukemia cells in promoting the growth, migration and invasion of B-ALL cells.Results: Compared with untreated MSCs, TGF-β conditioned MSCs significantly promoted the growth and proliferation of leukemia cells in mice, and increased the expression of CXCR4 in tumor tissues. In vitro cell experiments, TGF-β conditioned MSCs obviously promoted the migration and invasion of Nalm-6/RS4;11 cells, which were effectively blocked by the CXCR4 inhibitor AMD3100, thereby inhibiting the secretion of MMP-9 in TGF-β conditioned MSCs and inhibiting the activation of the PI3K/AKT signaling pathway in leukemia cells. Further, findings were made that the interaction between TGF-β conditioned MSCs and leukemia cells were mediated by the interaction between the integrin receptor α5β1 on the surface of leukemia cells and the increased expression of fibronectin on TGF-β conditioned MSCs. AMD3100 could weaken such effect by reducing the expression of integrin α5β1 on leukemia cells. Further regulation of integrin β1 could effectively interfere with the interaction between TGF-β conditioned MSCs and leukemia cells.Conclusion: Mesenchymal stem cells with CAF-like phenotype could be a key factor in promoting the growth and invasion of B-ALL cells, and the SDF-1/CXCR4 axis might be a significant factor in mediating the communication of MSCs with CAF-like phenotype and leukemia cells. To prevent the progression of B-ALL cells, blocking the SDF-1/CXCR4 axis with AMD3100 or targeting integrin β1 might be a potential therapeutic strategy.

The induction of bone formation: From bone morphogenetic proteins to the transforming growth factor-β3 protein - Redundancy, pleiotropy and the induction of cementogenesis

This review proposes to translate organogenesis and the induction of bone formation by the recombinant human transforming growth factor-β3 (hTGF-β3 ) in the Chacma baboon Papio ursinus to periodontal tissue induction and regeneration. Naturally derived highly purified osteogenic proteins of the transforming growth factor-β (TGF-β) supergene family were implanted in Class II furcation defects of the first and second mandibular molars. Additional defects in P.ursinus were treated with recombinant human osteogenic protein-1 (hOP-1, also known as bone morphogeneticprotein-7, hBMP-7) and hBMP-2, singly or in binary applications. In different studies defects were also implanted with hTGF-β3singly or in binary application with hOP-1. Harvested specimens on day 60 and 180 were processed for undecalcified histology using tungsten-carbide knives mounted on Polycut sledge’ micro-tomes or the Exakt precision cutting and grinding system.Highly purified osteogenic proteins showed the induction of Sharpey’s fibres into newly formed cementoid with foci of mineralization. hOP-1 induced substantial cementogenesis whilst hBMP-2 preferentially induced alveolar bone. Intramuscular implantation of hTGF-β3 absorbed onto coral-derived macroporous bioreactors engineered large heterotopic multicellular bone organoids. Gene expression pathways by quantitative Reverse Transcription Polymerases Chain Reaction (qRT-PCR) show that the induction of bone is via several profiled BMPs and TGF-βs expressed upon implantation of hTGF-β3 recapitulating the synergistic induction of bone as shown by binary applications of low doses of hTGF-β1 and hTGF-β3with hOP-1. The rapid induction of bone by hTGF-β3 provides theframework for a paradigmatic shift from recombinanthBMPs to hTGF-β3 in clinical contexts, provocatively operational in periodontal tissue regeneration with substantial induction of cementogenesis in angiogenesis.

Bifunctional TGF-β trap/IL-15 protein complex elicits potent NK cell and CD8+ T cell immunity against solid tumors

Advances in immunostimulatory and anti-immunosuppressive therapeutics have revolutionized cancer treatment. However, novel immunotherapeutics with these dual functions are not frequently reported. Here we describe the creation of a heterodimeric bifunctional fusion molecule, HCW9218, constructed using our soluble tissue factor (TF)-based scaffold technology. This complex comprises extracellular domains of the human transforming growth factor-β (TGF-β) receptor II and a human interleukin-15 (IL-15)/IL-15 receptor α complex. HCW9218 can be readily expressed in CHO cells and purified using antibody-based affinity chromatography in a large-scale manufacturing setting. HCW9218 potently activates mouse natural killer (NK) cells and CD8+ Tcells invitro and invivo to enhance cell proliferation, metabolism, and antitumor cytotoxic activities. Similarly, human immune cells become activated with increased cytotoxicity following incubation with HCW9218. This fusion complex also exhibits TGF-β neutralizing activity invitro and sequesters plasma TGF-β invivo. In a syngeneic B16F10 melanoma model, HCW9218 displayed strong antitumor activity mediated by NK cells and CD8+ Tcells and increased their infiltration into tumors. Repeat-dose subcutaneous administration of HCW9218 was well tolerated by mice, with a half-life sufficient to provide long-lasting biological activity. Thus, HCW9218 may serve as a novel therapeutic to simultaneously provide immunostimulation and lessen immunosuppression associated with tumors.

The Molecular Mechanism of Scutellaria baicalensis Georgi Stems and Leaves Flavonoids in Promoting Neurogenesis and Improving Memory Impairment by the PI3K-AKT-CREB Signaling Pathway in Rats.

The aim of this study was to investigate the effect and molecular mechanism of Scutellaria baicalensis Georgi stems and leaves flavonoids (SSF) in promoting neurogenesis and improving memory impairment induced by the PI3K-AKT-CREB signaling pathway. Alzheimer's disease (AD) was induced in the male Wistar rats by intracerebroventricular injection of amyloid beta peptide 25-35 (Aβ25-35) in combination with aluminum trichloride (AlCl3) and recombinant human transforming growth factor-β1(RHTGF-β1) (composited Aβ). The Morris water maze was used to screen the successful establishment of the memory impairment model of rats. The screened successful model rats were randomly divided into a model group and three groups of three different doses of the drug (SSF). Rats in the drug group were treated with 35, 70, and 140 mg/kg of SSF for 43 days. The Eight-arm maze was used to measure the spatial learning and memory abilities of the rat, including working memory errors (WME) and reference memory errors (RME). Immunohistochemistry was used to detect the expression of BrdU, an indicator of neuronal proliferation, in the hippocampal gyrus of rats. The mRNA and protein expressions of TRKB, PI3K, AKT, P-AKT, and IGF2 in the PI3K-AKT-CREB signaling pathway in the hippocampus and cerebral cortex of the rats were determined by quantitative real-time PCR (qPCR) and Western blotting methods. Compared to the sham group, the spatial memory ability of rats with composited Aβ was decreased, the number of WME and RME (P < 0.01) was increased, the expression of BrdU protein (P < 0.01) in the hippocampal gyrus was reduced, the mRNA and protein expression levels of TRKB, AKT, and IGF2 (P < 0.01, P < 0.05) in the hippocampus and cerebral cortex were lowered, and the mRNA expression level of PI3K (P < 0.01) in the cerebral cortex and the protein expression level of PI3K (P < 0.01) in the hippocampus were augmented. However, compared to the model group, the three-doses of SSF improved memory disorder induced by composited Aβ, reduced the number of WME and RME, increased the expression of BrdU protein in the hippocampal gyrus, and differently regulated the mRNA and protein expressions in composited Aβ rats. SSF improved memory impairment and neurogenesis disorder induced by composited Aβ in rats by activating the PI3K-AKT-CREB signaling pathway and up-regulating the mRNA and protein expressions of TRKB, PI3K, AKT, CREB, and IGF2.

Liquid PRF Reduces the Inflammatory Response and Osteoclastogenesis in Murine Macrophages.

Macrophage activation and osteoclastogenesis are hallmarks of inflammatory osteolysis and may be targeted by the local application of liquid platelet-rich fibrin (PRF). Liquid PRF is produced by a hard spin of blood in the absence of clot activators and anticoagulants, thereby generating an upper platelet-poor plasma (PPP) layer, a cell-rich buffy coat layer (BC; termed concentrated-PRF or C-PRF), and the remaining red clot (RC) layer. Heating PPP has been shown to generate an albumin gel (Alb-gel) that when mixed back with C-PRF generates Alb-PRF having extended working properties when implanted in vivo. Evidence has demonstrated that traditional solid PRF holds a potent anti-inflammatory capacity and reduces osteoclastogenesis. Whether liquid PRF is capable of also suppressing an inflammatory response and the formation of osteoclasts remains open. In the present study, RAW 264.7 and primary macrophages were exposed to lipopolysaccharides (LPS), lactoferrin, and agonists of Toll-like receptors (TLR3 and TLR7) in the presence or absence of lysates prepared by freeze-thawing of liquid PPP, BC, Alb-gel, and RC. For osteoclastogenesis, primary macrophages were exposed to receptor activator of nuclear factor kappa B ligand (RANKL), macrophage colony-stimulating factor (M-CSF), and human transforming growth factor-β1 (TGF-β1) in the presence or absence of PPP, BC, Alb-gel, RC lysates and hemoglobin. We show here that it is mainly the lysates prepared from PPP and BC that consistently reduced the agonist-induced expression of interleukin 6 (IL6) and cyclooxygenase-2 (COX2) in macrophages, as determined by RT-PCR and immunoassay. With respect to osteoclastogenesis, lysates from PPP and BC but also from RC, similar to hemoglobin, reduced the expression of osteoclast marker genes tartrate-resistant acid phosphatase (TRAP) and cathepsin K, as well as TRAP histochemical staining. These findings suggest that liquid PRF holds a potent in vitro heat-sensitive anti-inflammatory activity in macrophages that goes along with an inhibition of osteoclastogenesis.