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1.
Tissue Eng Regen Med ; 17(3): 335-350, 2020 06.
Article in English | MEDLINE | ID: mdl-32451775

ABSTRACT

BACKGROUND: Human adipose tissue-derived stem cells (ADSCs) are attractive multipotent stem cell sources with therapeutic potential in various fields requiring repair and regeneration, such as acute and chronically damaged tissues. ADSC is suitable for cell-based therapy, but its use has been hampered due to poor survival after administration. Potential therapeutic use of ADSC requires mass production of cells through in vitro expansion. Many studies have consistently observed the tendency of senescence by mesenchymal stem cell (MSC) proliferation upon expansion. Hypoxia has been reported to improve stem cell proliferation and survival. METHODS: We investigated the effects of hypoxia pretreatment on ADCS proliferation, migration capacity, differentiation potential and cytokine production. We also analyzed the effects of vascular endothelial growth factor (VEGF) on osteogenic and chondrogenic differentiation of ADSCs by hypoxia pretreatment. RESULTS: Hypoxia pretreatment increased the proliferation of ADSCs by increasing VEGF levels. Interestingly, hypoxia pretreatment significantly increased chondrogenic differentiation but decreased osteogenic differentiation compared to normoxia. The osteogenic differentiation of ADSC was decreased by the addition of VEGF but increased by the depletion of VEGF. We have shown that hypoxia pretreatment increases the chondrogenic differentiation of ADSCs while reducing osteogenic differentiation in a VEGF-dependent manner. CONCLUSION: These results show that hypoxia pretreatment can provide useful information for studies that require selective inhibition of osteogenic differentiation, such as cartilage regeneration.


Subject(s)
Adipocytes/metabolism , Adipose Tissue/metabolism , Chondrocytes/metabolism , Hypoxia/metabolism , Hypoxia/therapy , Stem Cells/metabolism , Vascular Endothelial Growth Factor A/metabolism , Adipose Tissue/cytology , Cell Differentiation/drug effects , Cell Movement , Cell Proliferation/drug effects , Chondrogenesis/drug effects , Cytokines/metabolism , Gene Expression , Gene Expression Profiling , Humans , Multipotent Stem Cells/metabolism , Osteogenesis , Stem Cells/cytology
2.
Mol Oncol ; 12(3): 356-372, 2018 03.
Article in English | MEDLINE | ID: mdl-29316206

ABSTRACT

The C-type lectin-like domain of CLEC14a (CLEC14a-C-type lectin-like domain [CTLD]) is a key domain that mediates endothelial cell-cell contacts in angiogenesis. However, the role of CLEC14a-CTLD in pathological angiogenesis has not yet been clearly elucidated. In this study, through complementarity-determining region grafting, consecutive deglycosylation, and functional isolation, we generated a novel anti-angiogenic human monoclonal antibody that specifically targets CLEC14a-CTLD and that shows improved stability and homogeneity relative to the parental antibody. We found that this antibody directly inhibits CLEC14a-CTLD-mediated endothelial cell-cell contact and simultaneously downregulates expression of CLEC14a on the surface of endothelial cells. Using various in vitro and in vivo functional assays, we demonstrated that this antibody effectively suppresses vascular endothelial growth factor (VEGF)-dependent angiogenesis and tumor angiogenesis of SNU182 human hepatocellular carcinoma, CFPAC-1 human pancreatic cancer, and U87 human glioma cells. Furthermore, we also found that this antibody significantly inhibits tumor angiogenesis of HCT116 and bevacizumab-adapted HCT116 human colorectal cancer cells. These findings suggest that antibody targeting of CLEC14a-CTLD has the potential to suppress VEGF-dependent angiogenesis and tumor angiogenesis and that CLEC14a-CTLD may be a novel anti-angiogenic target for VEGF-dependent angiogenesis and tumor angiogenesis.


Subject(s)
Antibodies, Monoclonal/pharmacology , Cell Adhesion Molecules/metabolism , Immunoglobulin G/pharmacology , Lectins, C-Type/metabolism , Neovascularization, Pathologic/drug therapy , Neovascularization, Physiologic/drug effects , Vascular Endothelial Growth Factor A/metabolism , Animals , Antibodies, Monoclonal/immunology , Cell Adhesion Molecules/genetics , Cell Communication/drug effects , Cell Communication/immunology , Cell Line, Tumor , Female , HCT116 Cells , Human Umbilical Vein Endothelial Cells , Humans , Immunoglobulin G/immunology , Lectins, C-Type/genetics , Mice , Mice, Inbred BALB C , Mice, Nude , Neovascularization, Pathologic/immunology , Neovascularization, Physiologic/immunology , Vascular Endothelial Growth Factor A/genetics , Xenograft Model Antitumor Assays
3.
Spine (Phila Pa 1976) ; 38(2): E49-58, 2013 Jan 15.
Article in English | MEDLINE | ID: mdl-23124260

ABSTRACT

STUDY DESIGN: An in vitro study with bovine intervertebral disc (IVD) cells and an in vivo study with a rabbit disc degeneration model on the extracellular matrix metabolism by a biglycan-derived peptide (Peniel 2000; P2K). OBJECTIVE: To investigate the mechanism for P2K-induced increases in extracellular matrix and in vitro and in vivo effects of the peptide on IVD. SUMMARY OF BACKGROUND DATA: Transforming growth factor-ß (TGF-ß) has a functional versatility on the metabolism of IVD cells, suggesting that the regulation of TGF-ß signaling is important in IVD degeneration. P2K was explored by an in silico drug discovery strategy to regulate TGF-ß signaling. METHODS: The putative target of P2K was verified by Biacore 3000 analysis and affinity purification using biotin-P2K. A monolayer culture system of bovine IVD cells was used to demonstrate the mechanism underlying the anabolic effects of P2K. Smad signaling and extracellular matrix metabolism of the IVD cells were investigated by Western blot and reverse transcription-polymerase chain reaction, respectively. The in vivo effect of P2K on degenerated disc was investigated using a rabbit model of disc degeneration. In 14 New Zealand white rabbits, disc degeneration was induced by percutaneous annular punctures. After 4 weeks, 3 consecutive discs in the same animal were treated with 5% lactose or P2K per disc. Twelve weeks after the treatment, the regenerative activity in the disc was examined by radiography, magnetic resonance imaging, and biochemical and histological analyses. RESULTS: Direct binding of P2K to an active form of TGF-ß1 was shown. Type II collagen and aggrecan were increased in TGF-ß1/P2K-treated bovine IVD cells, compared with nontreated and TGF-ß1-treated cells.In in vivo analysis, a single injection of P2K increased the disc height (P < 0.001) on the radiographs and improved the magnetic resonance imaging grade (P < 0.05) compared with controls. Biochemical analysis, showed a significant increase in PG content because of P2K treatment (P < 0.05). Histological analysis using disc degeneration grades demonstrated improvement in P2K-treated discs (P <0.01). CONCLUSION: A novel peptide, P2K, regulating TGF-ß1 signaling had an anabolic effect on bovine IVD cells and rabbit degenerated discs. The results suggest that P2K has considerable potential as a treatment of degenerative disc disease.


Subject(s)
Anabolic Agents/pharmacology , Biglycan/pharmacology , Intervertebral Disc Degeneration/drug therapy , Intervertebral Disc/drug effects , Transforming Growth Factor beta1/metabolism , Aggrecans/metabolism , Anabolic Agents/administration & dosage , Animals , Biglycan/administration & dosage , Cattle , Cells, Cultured , Collagen Type II/metabolism , Computer Simulation , Disease Models, Animal , Extracellular Matrix/drug effects , Extracellular Matrix/genetics , Extracellular Matrix/metabolism , Injections, Intralesional , Intervertebral Disc/pathology , Intervertebral Disc/physiology , Intervertebral Disc Degeneration/pathology , Magnetic Resonance Imaging , Male , Rabbits , Regeneration/drug effects , Signal Transduction/drug effects , Smad Proteins/genetics , Smad Proteins/metabolism
4.
Mol Ther ; 20(1): 178-86, 2012 Jan.
Article in English | MEDLINE | ID: mdl-21952165

ABSTRACT

Mesenchymal stem cells (MSCs) are emerging as a promising immunotherapeutic, based largely on their overt suppression of T lymphocytes under inflammatory and autoimmune conditions. While paracrine cross-talk between MSCs and T cells has been well-studied, an intrinsic transcriptional switch that programs MSCs for immunomodulation has remained undefined. Here we show that bone marrow-derived MSCs require the transcriptional regulator Aire to suppress T cell-mediated pathogenesis in a mouse model of chronic colitis. Surprisingly, Aire did not control MSC suppression of T cell proliferation in vitro. Instead, Aire reduced T cell mitochondrial reductase by negatively regulating a proinflammatory cytokine, early T cell activation factor (Eta)-1. Neutralization of Eta-1 enabled Aire(-/-) MSCs to ameliorate colitis, reducing the number of infiltrating effector T cells in the colon, and normalizing T cell reductase levels. We propose that Aire represents an early molecular switch imposing a suppressive MSC phenotype via regulation of Eta-1. Monitoring Aire expression in MSCs may thus be a critical parameter for clinical use.


Subject(s)
Crohn Disease/metabolism , Mesenchymal Stem Cells/metabolism , Transcription Factors/metabolism , Animals , Coculture Techniques , Crohn Disease/genetics , Crohn Disease/immunology , Female , Humans , Immunosuppression Therapy , Inflammation/immunology , Inflammation/metabolism , Intestinal Mucosa/metabolism , Intestines/immunology , Lymphocyte Activation/immunology , Mice , Mice, Inbred C57BL , Mice, Knockout , Osteopontin/genetics , Osteopontin/metabolism , Oxidation-Reduction , T-Lymphocytes/immunology , Transcription Factors/genetics , Transcription, Genetic , AIRE Protein
5.
Adv Exp Med Biol ; 633: 113-27, 2009.
Article in English | MEDLINE | ID: mdl-19209685

ABSTRACT

In this review we have highlighted the role of LNSCs in the regulation of CD8+ T cell immune responses in peripheral lymph nodes, thereby adding another layer of protection, in addition to the role of resting DCs, against autoimmunity. LNSCs have recently been implicated in the induction of peripheral CD8+ T cell tolerance due to their ability to endogenously express, process, and present PTAs. Furthermore, LNSCs express surface molecules, such as MHC class II and PD-L1, similar to those expressed by mTECs in the thymus and APCs. For future studies it will be important to address some of the new questions that have emerged with respect to the biology and function of LNSCs. Further work will help us to (1) dissect the specific roles that DCs and LNSCs have in the induction and maintenance of tolerance to intestinal antigens, (2) gain a more in-depth understanding of the molecular mechanisms underlying self-tolerance induction by LNSCs and the impact of inflammation on this function, (3) evaluate the relationship of LNSCs to the FRN, and (4) determine if the APC function of LNSCs extends to the acquisition and presentation of exogenous antigens. Finally, it is important to mention that so far the studies done on LNSCs have focused on their role in CD8+ T cell tolerance. At the moment, we do not know if presentation of PTAs by LNSCs can also induce tolerance of CD4+ T cells. Based on the finding that LNSCs express MHC class II (I-A(b)) molecules it is possible that they may present self-antigens to CD4+ T cells and induce tolerance. However, this has yet to be elucidated.


Subject(s)
Immune Tolerance/immunology , Lymph Nodes/cytology , Lymph Nodes/immunology , Stromal Cells/immunology , Animals , Humans , Intestines/immunology , Self Tolerance/immunology
6.
Gastroenterology ; 134(4): 1028-37, 2008 Apr.
Article in English | MEDLINE | ID: mdl-18395084

ABSTRACT

BACKGROUND & AIMS: Disruption of the enteric glial cell (EGC) network is an early pathologic feature in Crohn's disease. To determine the contribution of antigen-specific CD8 and CD4 T cells to the breakdown of the EGC network, we studied specific autoimmune targeting of an ectopic antigen expressed by EGCs. METHODS: Transgenic mice (GFAP-HA), which express the influenza hemagglutinin (HA) in EGCs, were either crossed with mice transgenic for a T-cell receptor (TCR) specific for a major histocompatibility complex (MHC) class I epitope of HA (CL4-TCR) or were adoptively transferred with conventional CL4 T cells. These were compared with GFAP-HA mice transferred with conventional T cells specific for an MHC class II epitope of HA (6.5). RESULTS: Both CD8 and CD4 T-cell subtypes were activated in vivo in an antigen-specific manner; however, they differed substantially in their ability to expand in the mesenteric lymph nodes, trigger proinflammatory cytokines, and induce autoimmune damage in the intestine. Direct presentation of antigen, provided by lymph node stromal cells, caused the activation and deletion of CD8 T cells. This mechanism of T-cell tolerance did not affect CD4 T cells, which produced antigen-specific lethal autoimmunity. CONCLUSIONS: Our observations support a recently identified mechanism of peripheral T-cell tolerance that specifically protects against autoimmunity mediated by conventional CD8 T cells. Furthermore, we show that conventional CD4 T cells are not affected by this mechanism of tolerance, and their targeting of EGCs produces lethal intestinal autoimmunity.


Subject(s)
Autoimmunity/immunology , CD8-Positive T-Lymphocytes/immunology , Enteritis/immunology , Lymph Nodes/immunology , Stromal Cells/immunology , Animals , Animals, Newborn , CD4-Positive T-Lymphocytes/immunology , Cells, Cultured , Cytokines/metabolism , Disease Models, Animal , Enteritis/pathology , Flow Cytometry , Gene Expression Regulation , Genes, MHC Class I , Histocompatibility Antigens Class I/biosynthesis , Histocompatibility Antigens Class I/genetics , Intestinal Mucosa/immunology , Intestinal Mucosa/pathology , Lymph Nodes/pathology , Lymphocyte Activation , Mesentery , Mice , Mice, Inbred BALB C , Mice, Transgenic , Minor Histocompatibility Antigens , RNA/genetics , Reverse Transcriptase Polymerase Chain Reaction , Stromal Cells/pathology
7.
Nat Immunol ; 8(2): 181-90, 2007 Feb.
Article in English | MEDLINE | ID: mdl-17195844

ABSTRACT

The intestinal epithelium functions to absorb nutrients and to protect the organism against microbes. To prevent autoimmune attack on this vital tissue, T cell tolerance to intestinal self-antigens must be established. Central tolerance mechanisms involve medullary thymic epithelial cells (mTECs), which use endogenously expressed peripheral-tissue antigens (PTAs) to delete self-reactive thymocytes. The prevailing model for the induction of peripheral tolerance involves cross-presentation of tissue antigens by quiescent dendritic cells. Here we show that lymph node stromal cells present endogenously expressed PTAs to T cells. Moreover, antigen presentation by lymph node stroma is sufficient to induce primary activation and subsequent tolerance among CD8(+) T cells. Thus, lymph node stromal cells are functionally akin to mTECs and provide a direct strategy for purging the peripheral repertoire of self-reactive T cells.


Subject(s)
Autoantigens/immunology , Intestines/immunology , Lymph Nodes/immunology , Self Tolerance/immunology , Stromal Cells/immunology , T-Lymphocytes/immunology , Animals , Autoantigens/genetics , Autoantigens/metabolism , Cell Proliferation , Dendritic Cells/immunology , Gene Expression Regulation , Intestinal Mucosa/metabolism , Lymph Nodes/cytology , Lymph Nodes/metabolism , Mice , Mice, Knockout , Stromal Cells/metabolism , T-Lymphocytes/cytology , T-Lymphocytes/metabolism
8.
Mol Cell Biol ; 26(6): 2309-16, 2006 Mar.
Article in English | MEDLINE | ID: mdl-16508006

ABSTRACT

Cathepsin F (cat F) is a widely expressed lysosomal cysteine protease whose in vivo role is unknown. To address this issue, mice deficient in cat F were generated via homologous recombination. Although cat F-/- mice appeared healthy and reproduced normally, they developed progressive hind leg weakness and decline in motor coordination at 12 to 16 months of age, followed by significant weight loss and death within 6 months. cat F was found to be expressed throughout the central nervous system (CNS). cat F-/- neurons accumulated eosinophilic granules that had features typical of lysosomal lipofuscin by electron microscopy. Large amounts of autofluorescent lipofuscin, characteristic of the neurodegenerative disease neuronal ceroid lipofuscinosis (NCL), accumulated throughout the CNS but not in visceral organs, beginning as early as 6 weeks of age. Pronounced gliosis, an indicator of neuronal stress and neurodegeneration, was also apparent in older cat F-/- mice. cat F is the only cysteine cathepsin whose inactivation alone causes a lysosomal storage defect and progressive neurological features in mice. The late onset suggests that this gene may be a candidate for adult-onset NCL.


Subject(s)
Cathepsins/deficiency , Nervous System Diseases/etiology , Neuronal Ceroid-Lipofuscinoses/etiology , Age Factors , Age of Onset , Animals , Cathepsin F , Cathepsins/genetics , Cathepsins/metabolism , Central Nervous System/metabolism , Central Nervous System/pathology , Lipofuscin/metabolism , Mice , Mice, Mutant Strains , Motor Neurons/pathology , Motor Neurons/ultrastructure , Neuromuscular Diseases/etiology , Neuronal Ceroid-Lipofuscinoses/epidemiology , Neuronal Ceroid-Lipofuscinoses/pathology , Neurons/pathology , Sequence Analysis, DNA , Spinal Cord/pathology , Spinal Cord/ultrastructure , Weight Loss
9.
Proc Natl Acad Sci U S A ; 102(49): 17729-33, 2005 Dec 06.
Article in English | MEDLINE | ID: mdl-16317068

ABSTRACT

The autoimmune cascade that culminates in diabetes initiates within pancreatic lymph nodes (PLNs). Here, we show that developmentally controlled lymphogenesis establishes a preferential trafficking route from the gut to the PLN, where T cells can be activated by antigens drained from the peritoneum and the gastrointestinal tract. Furthermore, intestinal stress modifies the presentation of pancreatic self-antigens in PLNs. The convergence of endocrine and intestinal contents within PLNs has significant implications for type 1 diabetes and may help to explain the link between autoimmune pathogenesis and environmental provocation.


Subject(s)
Endocrine System/immunology , Endocrine System/metabolism , Lymph Nodes/immunology , Lymph Nodes/metabolism , Pancreas/immunology , Pancreas/metabolism , Animals , Antigen Presentation/immunology , Antigens/immunology , Autoimmunity/immunology , CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/immunology , Cell Movement , Endocrine System/cytology , Lymph Nodes/cytology , Mice , Pancreas/cytology
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