ABSTRACT
Umbilical cord blood contains mesenchymal stem/stromal cells (MSCs) in addition to hematopoietic stem cells, serving as an attractive tool for regenerative medicine. As umbilical cord blood originates from fetus, abundant MSCs are expected to circulate in fetus. However, the properties of circulating MSCs in fetus have not been fully examined. In the present study, we aimed to analyze circulating MSCs, marked by the expression of platelet-derived growth factor receptor α (PDGFRα), during fetal development. Using PDGFRα GFP knock-in mice, we quantified the number of circulating PDGFRα positive MSCs during development. We further performed whole transcriptome analysis of circulating MSCs at single cell levels. We found that abundant PDGFRα positive cells circulate in embryo and diminish immediately after birth. In addition, single cell RNA-sequencing revealed transcriptional heterogeneity of MSCs in fetal circulation. These data lay a foundation to analyze the function of circulating MSCs during development.
Subject(s)
Fetal Blood/cytology , Fetal Blood/metabolism , Fetus/cytology , Fetus/metabolism , Mesenchymal Stem Cells/cytology , Mesenchymal Stem Cells/metabolism , Animals , Cell Count , Female , Mice , Mice, Inbred C57BL , Mice, Transgenic , Pregnancy , Receptor, Platelet-Derived Growth Factor alpha/genetics , Receptor, Platelet-Derived Growth Factor alpha/metabolism , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Regenerative Medicine , Single-Cell Analysis , Transcription, GeneticABSTRACT
Recessive dystrophic epidermolysis bullosa (RDEB) is an intractable genetic blistering skin disease in which the epithelial structure easily separates from the underlying dermis because of genetic loss of functional type VII collagen (Col7) in the cutaneous basement membrane zone. Recent studies have demonstrated that allogeneic bone marrow transplantation (BMT) ameliorates the skin blistering phenotype of RDEB patients by restoring Col7. However, the exact therapeutic mechanism of BMT in RDEB remains unclear. In this study, we investigated the roles of transplanted bone marrow-derived circulating mesenchymal cells in RDEB (Col7-null) mice. In wild-type mice with prior GFP-BMT after lethal irradiation, lineage-negative/GFP-positive (Lin(-)/GFP(+)) cells, including platelet-derived growth factor receptor α-positive (PDGFRα(+)) mesenchymal cells, specifically migrated to skin grafts from RDEB mice and expressed Col7. Vascular endothelial cells and follicular keratinocytes in the deep dermis of the skin grafts expressed SDF-1α, and the bone marrow-derived PDGFRα(+) cells expressed CXCR4 on their surface. Systemic administration of the CXCR4 antagonist AMD3100 markedly decreased the migration of bone marrow-derived PDGFRα(+) cells into the skin graft, resulting in persistent epidermal detachment with massive necrosis and inflammation in the skin graft of RDEB mice; without AMD3100 administration, Col7 was significantly supplemented to ameliorate the pathogenic blistering phenotype. Collectively, these data suggest that the SDF1α/CXCR4 signaling axis induces transplanted bone marrow-derived circulating PDGFRα(+) mesenchymal cells to migrate and supply functional Col7 to regenerate RDEB skin.
Subject(s)
Bone Marrow Transplantation , Collagen Type VII/metabolism , Epidermolysis Bullosa Dystrophica/metabolism , Epidermolysis Bullosa Dystrophica/pathology , Mesenchymal Stem Cell Transplantation , Animals , Cell Separation , Chemokine CXCL12/metabolism , Disease Models, Animal , Enzyme-Linked Immunosorbent Assay , Flow Cytometry , Fluorescent Antibody Technique , Mice , Mice, Inbred C57BL , Mice, Knockout , Real-Time Polymerase Chain Reaction , Receptor, Platelet-Derived Growth Factor alpha/metabolism , Receptors, CXCR4/metabolism , Signal Transduction/physiology , Skin TransplantationABSTRACT
The physiological role of "endogenous" bone marrow (BM) mesenchymal stromal cells (MSCs) in tissue regeneration is poorly understood. Here, we show the significant contribution of unique endogenous BM-MSC populations to muscle regeneration in Duchenne muscular dystrophy (DMD) mice (mdx). Transplantation of BM cells (BMCs) from 10-week-old mdx into 3-4-week-old mdx mice increased inflammation and fibrosis and reduced muscle function compared with mdx mice that received BMCs from 10-week-old wild-type mice, suggesting that the alteration of BMC populations in mdx mice affects the progression of muscle pathology. Two distinct MSC populations in BM, that is, hematopoietic lineage (Lin)(-) /ckit(-) /CD106(+) /CD44(+) and Lin(-) /ckit(-) /CD106(+) /CD44(-) cells, were significantly reduced in 10-week-old mdx mice in disease progression. The results of a whole-transcriptome analysis indicated that these two MSC populations have distinct gene expression profiles, indicating that the Lin(-) /ckit(-) /CD106(+) /CD44(+) and Lin(-) /ckit(-) /CD106(+) /CD44(-) MSC populations are proliferative- and dormant-state populations in BM, respectively. BM-derived Lin(-) /CD106(+) /CD44(+) MSCs abundantly migrated to damaged muscles and highly expressed tumor necrosis factor-alpha-stimulated gene/protein-6 (TSG-6), an anti-inflammatory protein, in damaged muscles. We also demonstrated that TSG-6 stimulated myoblast proliferation. The injection of Lin(-) /ckit(-) /CD106(+) /CD44(+) MSCs into the muscle of mdx mice successfully ameliorated muscle dysfunction by decreasing inflammation and enhancing muscle regeneration through TSG-6-mediated activities. Thus, we propose a novel function of the unique endogenous BM-MSC population, which countered muscle pathology progression in a DMD model.
Subject(s)
Bone Marrow Cells/physiology , Mesenchymal Stem Cells/physiology , Muscles/pathology , Muscular Diseases/pathology , Animals , Bone Marrow Cells/metabolism , Cell Movement/physiology , Cell Proliferation/physiology , Cells, Cultured , Mesenchymal Stem Cells/metabolism , Mice , Mice, Inbred C57BL , Mice, Inbred mdx , Muscular Diseases/metabolismABSTRACT
The role of bone marrow cells in repairing ectodermal tissue, such as skin epidermis, is not clear. To explore this process further, this study examined a particular form of cutaneous repair, skin grafting. Grafting of full thickness wild-type mouse skin onto mice that had received a green fluorescent protein-bone marrow transplant after whole body irradiation led to an abundance of bone marrow-derived epithelial cells in follicular and interfollicular epidermis that persisted for at least 5 mo. The source of the epithelial progenitors was the nonhematopoietic, platelet-derived growth factor receptor α-positive (Lin(-)/PDGFRα(+)) bone marrow cell population. Skin grafts release high mobility group box 1 (HMGB1) in vitro and in vivo, which can mobilize the Lin(-)/PDGFRα(+) cells from bone marrow to target the engrafted skin. These data provide unique insight into how skin grafts facilitate tissue repair and identify strategies germane to regenerative medicine for skin and, perhaps, other ectodermal defects or diseases.
Subject(s)
Bone Marrow Cells/metabolism , Epidermis/injuries , Epidermis/metabolism , HMGB1 Protein/metabolism , Receptor, Platelet-Derived Growth Factor alpha/metabolism , Regeneration , Animals , Bone Marrow Transplantation , Graft Survival/genetics , HMGB1 Protein/genetics , Mice , Mice, Transgenic , Receptor, Platelet-Derived Growth Factor alpha/genetics , Skin Transplantation , Transplantation, HomologousABSTRACT
Recessive dystrophic epidermolysis bullosa (RDEB) is a genodermatosis caused by variants in COL7A1-encoded type VII collagen, a major component of anchoring fibrils. In this study, we developed an ex vivo gene therapy for RDEB using autologous mesenchymal stromal cells (MSCs). On the basis of our previous studies, we first attempted to isolate MSCs from the blister fluid of patients with RDEB and succeeded in obtaining cells with a set of MSC characteristics from all 10 patients. We termed these cells blister fluid-derived MSCs. Blister fluid-derived MSCs were genetically modified and injected into skins of type VII collagen-deficient neonatal mice transplanted onto immunodeficient mice, resulting in continuous and widespread expression of type VII collagen at the dermal-epidermal junction, particularly when administered into blisters. When injected intradermally, the efforts were not successful. The gene-modified blister fluid-derived MSCs could be cultured as cell sheets and applied to the dermis with an efficacy equivalent to that of intrablister administration. In conclusion, we successfully developed a minimally invasive and highly efficient ex vivo gene therapy for RDEB. This study shows the successful application of gene therapy in the RDEB mouse model for both early blistering skin and advanced ulcerative lesions.
Subject(s)
Epidermolysis Bullosa Dystrophica , Mesenchymal Stem Cells , Humans , Mice , Animals , Epidermolysis Bullosa Dystrophica/genetics , Epidermolysis Bullosa Dystrophica/therapy , Epidermolysis Bullosa Dystrophica/pathology , Blister/genetics , Blister/therapy , Collagen Type VII/genetics , Collagen Type VII/metabolism , Skin/pathology , Genes, Recessive , Mesenchymal Stem Cells/metabolismABSTRACT
Malignant glioma is one of the most aggressive cancers. For the development of effective therapeutic strategies against such malignant diseases, elucidation of molecular targets is necessary. We found that inactivated Sendai virus particle (HVJ-E) induced extensive cell death in the human glioblastoma cell line U251MG. Intradermal U251MG tumors were more effectively suppressed by HVJ-E than interferon (IFN)-beta. From microarray analysis of gene expression in U251MG cells treated with HVJ-E, we focused on the up-regulation of sterile alpha motif containing domain 9 (SAMD9) gene. The expression of the SAMD9 gene was induced by administration of recombinant human IFN-alpha, -beta or -gamma. The up-regulation of the SAMD9 gene by HVJ-E treatment was abrogated by IFN receptor blocking antibody or JAK inhibitor treatment. When SAMD9 expression was knocked down by RNA interference, apoptotic cell death induced by HVJ-E was blocked in U251MG cells. Suppression of SAMD9 using SAMD9 siRNA also inhibited IFN-beta-induced death in U251MG cells with a small, but significant, difference to control groups. However, overexpression of the SAMD9 gene failed to induce significant cell death in U251MG cells. Thus, SAMD9 could be a key molecule to control cancer cell death by HVJ-E or IFN-beta treatment.
Subject(s)
Antineoplastic Agents/pharmacology , Glioma/genetics , Glioma/therapy , Interferon Type I/pharmacology , Oncolytic Virotherapy/methods , Proteins/genetics , Animals , Apoptosis/physiology , Blotting, Western , Cell Line, Tumor , Cell Separation , Enzyme-Linked Immunosorbent Assay , Flow Cytometry , Humans , Interferon Type I/metabolism , Intracellular Signaling Peptides and Proteins , Male , Mice , Mice, SCID , Oligonucleotide Array Sequence Analysis , Proteins/metabolism , RNA Interference , Recombinant Proteins/metabolism , Recombinant Proteins/pharmacology , Reverse Transcriptase Polymerase Chain Reaction , Sendai virus/immunology , Virion/immunology , Virus Inactivation , Xenograft Model Antitumor AssaysABSTRACT
Bacterial attachment and growth on material surfaces are considered to be the primary steps leading to the formation of biofilm. Biofilms in hospital and food processing settings can result in bacterial infection and food contamination, respectively. Prevention of bacterial attachment, therefore, is considered to be the best strategy for abating these menaces and therefore the development of antibacterial metals becomes important. In this study, nine pure metals, viz. titanium, cobalt, nickel, copper, zinc, zirconium, molybdenum, tin, and lead have been tested for their antibacterial properties against two bacterial strains, Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli. This was accomplished using two assay methods, the film contact method and the shaking flask method. The results show that the antibacterial properties varied significantly with different metals and the effectiveness of metals to resist bacterial attachment varied with the bacterial strain. Among the metals tested, titanium and tin did not exhibit antibacterial properties. TEM images showed that metal accumulation resulted in the disruption of the bacterial cell wall and other cellular components.
Subject(s)
Anti-Bacterial Agents/pharmacology , Escherichia coli/drug effects , Metals, Heavy/pharmacology , Staphylococcus aureus/drug effects , Bacterial Adhesion/drug effects , Biofilms/drug effects , Biofilms/growth & development , Cell Wall/drug effects , Culture Media , Escherichia coli/growth & development , Escherichia coli/ultrastructure , Microbial Sensitivity Tests/methods , Microscopy, Electron, Transmission , Staphylococcus aureus/growth & development , Staphylococcus aureus/ultrastructureABSTRACT
The utility of various synthetic peptides has been investigated in clinical trials of the treatment of cancers, infectious diseases and endocrine diseases. In the process of functional gene screening with in silico analysis for molecules with angiogenic properties, we generated a small peptide, angiogenic peptide (AG)-30, that possesses both antimicrobial and pro-inflammatory activities. AG-30 has an alpha-helix structure with a number of hydrophobic or net positively charged amino acids and a propensity to fold into amphipathic structures. Indeed, AG-30 exhibited antimicrobial activity against various bacteria, induced vascular endothelial cell growth and tube formation in a dose-dependent manner and increased neovascularization in a Matrigel plug assay. As a result, AG-30 up-regulated expression of angiogenesis-related cytokines and growth factors for up to 72 hrs in human aortic endothelial cells. To further evaluate the angiogenic effect of AG-30 in vivo, we developed a slow-release AG-30 system utilizing biodegradable gelatin microspheres. In the ischaemic mouse hind limb, slow-release AG-30 treatment results in an increase in angiogenic score, an increase in blood flow (as demonstrated by laser Doppler imaging) and an increase in capillary density (as demonstrated by immunostaining with anti-CD31 antibody). These data suggest that the novel peptide, AG-30, may have therapeutic potential for ischaemic diseases.
Subject(s)
Antimicrobial Cationic Peptides/chemical synthesis , Antimicrobial Cationic Peptides/pharmacology , Neovascularization, Physiologic/drug effects , Amino Acid Sequence , Animals , Antimicrobial Cationic Peptides/chemistry , Bacteria/drug effects , Cells, Cultured , Collagen/metabolism , Delayed-Action Preparations , Disease Models, Animal , Drug Combinations , Endothelial Cells/drug effects , Hindlimb/blood supply , Hindlimb/drug effects , Humans , Ischemia/chemically induced , Ischemia/therapy , Laminin/metabolism , Mice , Mice, Inbred C57BL , Microbial Sensitivity Tests , Molecular Sequence Data , Oligonucleotide Array Sequence Analysis , Protein Conformation , Proteoglycans/metabolism , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
Recent studies have shown that skin injury recruits bone marrow-derived fibroblasts (BMDFs) to the site of injury to accelerate tissue repair. However, whether uninjured skin can recruit BMDFs to maintain skin homeostasis remains uncertain. Here, we investigated the appearance of BMDFs in normal mouse skin after embryonic bone marrow cell transplantation (E-BMT) with green fluorescent protein-transgenic bone marrow cells (GFP-BMCs) via the vitelline vein, which traverses the uterine wall and is connected to the fetal circulation. At 12 weeks of age, mice treated with E-BMT were observed to have successful engraftment of GFP-BMCs in hematopoietic tissues accompanied by induction of immune tolerance against GFP. We then investigated BMDFs in the skin of the same mice without prior injury and found that a significant number of BMDFs, which generate matrix proteins both in vitro and in vivo, were recruited and maintained after birth. Next, we performed E-BMT in a dystrophic epidermolysis bullosa mouse model (col7a1(-/-)) lacking type VII collagen in the cutaneous basement membrane zone. E-BMT significantly ameliorated the severity of the dystrophic epidermolysis bullosa phenotype in neonatal mice. Type VII collagen was deposited primarily in the follicular basement membrane zone in the vicinity of the BMDFs. Thus, gene therapy using E-BMT into the fetal circulation may offer a potential treatment option to ameliorate genetic skin diseases that are characterized by fibroblast dysfunction through the introduction of immune-tolerated BMDFs.
Subject(s)
Bone Marrow Transplantation , Epidermolysis Bullosa Dystrophica/therapy , Fibroblasts/cytology , Genetic Therapy/methods , Immune Tolerance , Skin/cytology , Animals , Bone Marrow Transplantation/methods , Collagen Type VII/metabolism , Embryonic Stem Cells/transplantation , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/immunology , Mice , Mice, Inbred C57BL , Mice, Transgenic , Microscopy, Confocal , Microscopy, Fluorescence , Reverse Transcriptase Polymerase Chain Reaction , Skin/immunologyABSTRACT
The possibility of using bacteria to drill metallic surfaces has been demonstrated using Staphylococcus sp., a facultative anaerobic bacterium, isolated from corroded copper piping. The experiment involved exposure of copper coupons (25 mm x 15 mm x 3 mm) to a culture of Staphylococcus sp. for a maximum period of 7 days. Coupons exposed to sterile bacterial growth medium were used as controls. Exposed coupons were removed intermittently and observed microscopically for the extent of drilling. The total pit area and volume on these coupons were determined using image analysis. The results showed that both the biomachined area and volume increased with the duration of coupon exposure. In the drilling experiment, a copper thin film 2 microm thick was perforated by this bacterium within a period of 7 days. In conclusion, the results suggested that bacteria can be used as a tool for machining metallic surfaces.
Subject(s)
Copper/metabolism , Staphylococcus/metabolism , Ammonia/metabolism , Bacteria, Anaerobic , Biofilms , Chlorides/chemistry , Corrosion , Electric Conductivity , Equipment Design , Fermentation , Fresh Water/microbiology , Hydrogen-Ion Concentration , Lactic Acid/biosynthesis , Microscopy, Confocal , Microscopy, Electron, Scanning , Models, Biological , RNA, Ribosomal, 16S , Sequence Alignment , Sequence Analysis, RNA , Staphylococcus/isolation & purification , Substrate Specificity , Temperature , Time FactorsABSTRACT
Over the past decade, many approaches to transferring genes into the skin have been investigated. However, most such approaches have been specifically aimed against genodermatosis, and have not produced sufficient results. The goal of such research is to develop a method in which genes are transferred easily, efficiently and stably into keratinocytes, especially into keratinocyte stem cells, and in which the transgene expression persists without a reaction from the host immune response. Although accidental development of cancer has occurred in trials of gene therapy for X-linked severe combined immunodeficiency (X-SCID), resulting in slowing of the progress of this research, the lessons of these setbacks have been applied to further research. Moreover, combined with the techniques acquired from tissue engineering, recent developments in our knowledge about stem cells will lead to new treatments for genodermatoses. The present review summarizes the methods by which therapeutic genes can be transferred into keratinocytes, with discussion of how gene transfer efficiency can be improved, with particular emphasis on disruption of the skin barrier function. It concludes with discussion of the challenges and prospects of keratinocyte gene therapy, in terms of achieving efficient and long-lasting therapeutic effects.
Subject(s)
DNA/metabolism , Gene Transfer Techniques , Genetic Therapy/methods , Skin Absorption , Skin Diseases, Genetic/therapy , Skin/metabolism , Animals , Genetic Therapy/adverse effects , Humans , Keratinocytes/metabolism , Permeability , Skin Diseases, Genetic/genetics , Skin Diseases, Genetic/metabolismABSTRACT
OBJECTIVE: We identified a ubiquitin carboxyl-terminal hydrolase L1 (UCHL1) gene, which encodes a deubiquitinating enzyme and is expressed in the vasculature, by functional screening of a human endothelial cell (EC) cDNA library. UCHL1 is expressed in neurons, and abnormalities in UCHL1 are responsible for inherited Parkinson's disease via its effects on the ubiquitin-proteasome system. Therefore, the goal of present study was to clarify the role of the UCHL1 gene in vascular remodeling by evaluating nuclear factor-kappaB (NF-kappaB) inactivation in ECs and vascular smooth muscle cells (VSMCs). METHODS AND RESULTS: From Northern blot and immunohistochemical analysis, the UCHL1 gene was endogenously expressed in vascular ECs, VSMCs, and brain tissue. Expression of UCHL1 was markedly increased in the neointima of the balloon-injured carotid artery and was also present in atherosclerotic lesions from human carotid arteries. Overexpression of the UCHL1 gene significantly attenuated tumor necrosis factor (TNF)-alpha-induced NF-kappaB activity in vascular cells and increased inhibitor of kappa B-alpha (IkappaB-alpha), possibly through the attenuation of IkappaB-alpha ubiquitination, leading to decreased neointima in the balloon-injured artery. In contrast, knockdown of UCHL1 by small interfering RNA resulted in increased NF-kappaB activity in VSMCs. CONCLUSIONS: These data suggest that UCHL1 may partially attenuate vascular remodeling through inhibition of NF-kappaB activity.
Subject(s)
Endothelial Cells/metabolism , I-kappa B Proteins/metabolism , Muscle, Smooth, Vascular/metabolism , NF-kappa B/metabolism , Protein Processing, Post-Translational , Ubiquitin Thiolesterase/metabolism , Ubiquitin/metabolism , Animals , Carotid Artery Injuries/enzymology , Carotid Artery Injuries/etiology , Carotid Artery Injuries/metabolism , Carotid Stenosis/enzymology , Catheterization/adverse effects , Cells, Cultured , Disease Models, Animal , Endothelial Cells/enzymology , Humans , Muscle, Smooth, Vascular/enzymology , Myocytes, Smooth Muscle/enzymology , Myocytes, Smooth Muscle/metabolism , NF-KappaB Inhibitor alpha , NF-kappa B/genetics , Nitric Oxide/metabolism , Nitric Oxide Synthase Type III/metabolism , RNA Interference , RNA, Messenger/metabolism , RNA, Small Interfering/metabolism , Rats , Time Factors , Transfection , Tumor Necrosis Factor-alpha/metabolism , Ubiquitin Thiolesterase/analysis , Ubiquitin Thiolesterase/genetics , Up-RegulationABSTRACT
Mesenchymal stem cells (MSCs), which can differentiate into tri-lineage (osteoblast, adipocyte, and chondrocyte) and suppress inflammation, are promising tools for regenerative medicine. MSCs are phenotypically diverse based on their tissue origins. However, the mechanisms underlying cell-type-specific gene expression patterns are not fully understood due to the lack of suitable strategy to identify the diversity. In this study, we investigated gene expression programs and chromatin accessibilities of MSCs by whole-transcriptome RNA-seq analysis and an assay for transposase-accessible chromatin using sequencing (ATAC-seq). We isolated MSCs from four tissues (femoral and vertebral bone marrow, adipose tissue, and lung) and analysed their molecular signatures. RNA-seq identified the expression of MSC markers and both RNA-seq and ATAC-seq successfully clustered the MSCs based on their tissue origins. Interestingly, clustering based on tissue origin was more accurate with chromatin accessibility signatures than with transcriptome profiles. Furthermore, we identified transcription factors potentially involved in establishing cell-type specific chromatin structures. Thus, epigenome analysis is useful to analyse MSC identity and can be utilized to characterize these cells for clinical use.
Subject(s)
Chromatin/genetics , Chromatin/metabolism , Mesenchymal Stem Cells/metabolism , Adipocytes/metabolism , Adipocytes/physiology , Adipose Tissue/metabolism , Adipose Tissue/physiology , Animals , Biomarkers/metabolism , Bone Marrow Cells/metabolism , Bone Marrow Cells/physiology , Cluster Analysis , Femur/metabolism , Femur/physiology , Gene Expression/genetics , Gene Expression/physiology , Gene Expression Profiling/methods , Lung/metabolism , Lung/physiology , Mice , Mice, Inbred C57BL , Transcription Factors/genetics , Transcription Factors/metabolism , Transcriptome/genetics , Transcriptome/physiologyABSTRACT
We report a novel strategy for constructing a tissue-targeting hemagglutinating virus of Japan (HVJ; Sendai virus) envelope vector (HVJ-E), and its application in gene therapy of a mouse model of genetic skin disease. Chimeric genes encoding viral F protein and green fluorescent protein (GFP) were constructed on the basis of various deletion mutants. The product of one chimeric gene, containing signal peptide, transmembrane domain, and the cytoplasmic tail of F protein, was transported to the cell surface and incorporated into new viruses released from HVJ-infected LLC-MK2 cells. For tissue targeting, in the preceding construct GFP was replaced with single-chain antibody (scFv) against mouse desmoglein 3 (mDsg3), a desmosomal cadherin found in basal layer keratinocytes of the skin. HVJ encoding scFv-F chimeric protein bound to mDsg3-coated plates much more efficiently than did wild-type HVJ. When chimeric HVJ was injected into a skin blister of a mouse model of epidermolysis bullosa, in which defective expression of type VII collagen results in a failure to secure epidermis to the underlying dermis, viral F protein expression was detected in most of the basal keratinocytes. Furthermore, chimeric HVJ-E introduced type VII collagen expression more efficiently compared with wild-type HVJ in basal keratinocytes of type VII collagen-deficient mouse skin, resulting in efficient amelioration of the genetic defect. Thus, a novel tissue-targeting HVJ-E could be used to successfully target epidermal keratinocytes both in vitro and in vivo.
Subject(s)
Desmoglein 3/metabolism , Epidermolysis Bullosa/therapy , Genetic Therapy , Genetic Vectors , Keratinocytes/metabolism , Sendai virus/genetics , Viral Fusion Proteins/metabolism , Animals , Cell Line , Epidermolysis Bullosa/genetics , Gene Targeting , Gene Transfer Techniques , Keratinocytes/cytology , Mice , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Viral Fusion Proteins/geneticsABSTRACT
BACKGROUND: Lymphedema is a disorder of the lymphatic vascular system characterized by impaired lymphatic return and swelling of the extremities. Treatment for this disabling condition remains limited and largely ineffective. The goal of the present study was to investigate the therapeutic efficacy of hepatocyte growth factor (HGF) in animal models of lymphedema. METHODS AND RESULTS: Immunofluorescent analysis demonstrated that canine primary lymphatic endothelial cells (cLECs) were positive for lymphatic-specific markers (vascular endothelial growth factor receptor-3, LYVE-1, podoplanin, and Prox1) and the HGF receptor c-Met. Treating cLECs with human recombinant HGF resulted in a dose-dependent increase in cell growth and migration and increased activity of extracellular signal-regulated kinase and Akt. In human LECs, c-Met also was expressed, and treatment with HGF increased cell growth and migration in a dose-dependent manner. Transfection of human HGF plasmid DNA in cLECs also increased the c-fos promoter activity. Furthermore, weekly HGF gene transfer in a rat tail lymphedema model by disruption of lymphatic vessels resulted in a decrease in lymphedema thickness. Although expression of the endothelial cell marker PECAM-1 was increased in both HGF- and vascular endothelial growth factor 165-injected groups, expression of LEC markers (LYVE-1 and Prox1) was increased only in the HGF-injected group. CONCLUSIONS: These data demonstrate that expression of HGF via plasmid transfer improves lymphedema via promotion of lymphangiogenesis. Further studies to determine the clinical utility of this approach would be of benefit to patients with lymphedema.
Subject(s)
Hepatocyte Growth Factor/genetics , Lymphangiogenesis/genetics , Lymphedema/genetics , Lymphedema/therapy , Transfection , Animals , Cell Movement/drug effects , Cell Proliferation/drug effects , Cells, Cultured , DNA/genetics , Disease Models, Animal , Dogs , Dose-Response Relationship, Drug , Endothelium, Vascular/cytology , Endothelium, Vascular/drug effects , Endothelium, Vascular/metabolism , Female , Gene Expression Regulation , Gene Transfer Techniques , Genetic Therapy , Hepatocyte Growth Factor/pharmacology , Hepatocyte Growth Factor/therapeutic use , Humans , Lymphangiogenesis/physiology , Lymphedema/pathology , Lymphedema/physiopathology , Male , Proto-Oncogene Proteins c-met/genetics , Proto-Oncogene Proteins c-met/metabolism , Rats , Rats, Sprague-DawleyABSTRACT
We examined a gene polymorphism of a novel Z-disc-related protein, myospryn (cardiomyopathy-associated 5). We focused on one haplotype block associated with a tag single nucleotide polymorphism (SNP) that covered 16 of 27 coding SNPs with linkage disequilibrium (minor allele frequency 0.413). Screening a myospryn polymorphism (K2906N) in a general health check-up of a rural Japanese population revealed an association with cardiac diseases (p=0.0082). In further analysis of the interaction between K2906N and cardiac function in patients, K2906N was associated with the anteroseptal wall thickness of the left ventricle in a recessive model (p=0.0324) and with the ratio of the peak velocity of the early diastolic filling wave to the peak velocity of atrial filling (A/E) (p=0.0278). In an association study based on left ventricular wall thickness, we found a significant difference in the K2906N genotype between controls and patients with cardiac hypertrophy. These results suggest that the K2906N polymorphism could be clinically associated with left ventricular hypertrophy and diastolic dysfunction independent of known parameters. Although the precise mechanism underlying this association remains to be elucidated, treatment with angiotensin II induced an increase in heart myospryn mRNA level in vitro and in vivo. Our results suggest that the polymorphism of myospryn is associated with left ventricular hypertrophy, and an association between a Z-disc protein and cardiac adaptation in response to pressure overload.
Subject(s)
Hypertension/complications , Hypertension/genetics , Hypertrophy, Left Ventricular/etiology , Hypertrophy, Left Ventricular/genetics , Muscle Proteins/genetics , Polymorphism, Single Nucleotide/genetics , Adult , Aged , Aged, 80 and over , Angiotensin II/pharmacology , Animals , Case-Control Studies , Cells, Cultured , Disease Models, Animal , Female , Heart Ventricles/diagnostic imaging , Heart Ventricles/metabolism , Heart Ventricles/pathology , Humans , Hypertrophy, Left Ventricular/pathology , Linkage Disequilibrium/genetics , Male , Mice , Mice, Inbred C57BL , Middle Aged , Muscle Proteins/metabolism , Myocytes, Cardiac/drug effects , Myocytes, Cardiac/metabolism , Myocytes, Cardiac/pathology , Rats , Rats, Sprague-Dawley , Ultrasonography , Vasoconstrictor Agents/pharmacologyABSTRACT
We investigated characteristics of the corrosion of stainless steel specimens by bacteria and the effects of using antimicrobial coating on the surface for inhibiting corrosion. Bacillus sp. 2-A and Staphylococcus sp. 2-1 cells adhered tightly to a stainless steel SUS304 specimen, formed a microcolony or biofilm, and had highly corrosive activities. Microbially influenced corrosion (MC) was observed under or around adhering cells. However, dead cells were markedly less active than viable cells not only in corroding the specimen but also in adhering to its surface. The culture supernatant was not able to induce the corrosion of SUS304 effectively. A protamine coating on the specimen killed bacterial cells only on its surface, interfered with cell adhesion, and inhibited MC. From these results, adhesion of viable cells to the surface of a SUS304 specimen led to the outbreak of MC. Protamine was also found to be an effective substance tested for protecting the specimen from both cell adhesion and surface MC. We suggest that a protamine coating can be applied as a convenient and inexpensive corrosion prevention method.
Subject(s)
Bacterial Adhesion/physiology , Corrosion , Protamines/chemistry , Stainless Steel , Adhesiveness , Bacillus cereus/growth & development , Bacillus cereus/isolation & purification , Bacillus cereus/physiology , DNA, Bacterial/analysis , DNA, Ribosomal/analysis , Microscopy, Electron, Scanning , RNA, Bacterial/analysis , RNA, Ribosomal, 16S/analysis , Stainless Steel/chemistry , Stainless Steel/standards , Staphylococcus/growth & development , Staphylococcus/isolation & purification , Staphylococcus/physiologyABSTRACT
Adult stem cells hold great promise for use in tissue repair and regeneration, and the delivery of autologous progenitor cells into ischemic tissue is emerging as a novel therapeutic option. We and others have recently demonstrated the potential impact of adipose tissue-derived stromal cells (ADSC) on regenerative cell therapy for ischemic diseases. The main benefit of ADSC is that they can be easily harvested from patients by a simple, minimally invasive method and also easily cultured. Cultured ADSC can be induced to differentiate into not only adipocytes, but also bone, neurons or endothelial cells in certain conditions. Interestingly, they secrete a number of angiogenesis-related cytokines, such as vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF), which might be suitable for regenerative cell therapy for ischemic diseases. In the ischemic mouse hindlimb, the angiogenic score was improved in the ADSC-treated group. Moreover, recent reports demonstrated that these ADSC can also be induced to differentiate into cardiac myocytes. These adipose tissue-derived cells have potential in angiogenic cell therapy for ischemic disease, and might be applied for regenerative cell therapy instead of bone marrow cells in the near future.
Subject(s)
Adipose Tissue/cytology , Stromal Cells/cytology , Stromal Cells/transplantation , Animals , Cell Differentiation , Humans , Mesenchymal Stem Cell Transplantation , Mesenchymal Stem Cells/cytology , Mice , Models, Animal , Neovascularization, Physiologic , Regeneration , Transplantation, AutologousABSTRACT
OBJECTIVE: The delivery of autologous progenitor cells into ischemic tissue of patients is emerging as a novel therapeutic option. Here, we report the potential impact of cultured adipose tissue-derived cells (ADSC) on angiogenic cell therapy. METHOD AND RESULTS: ADSC were isolated from C57Bl/6 mouse inguinal adipose tissue and showed high expression of ScaI and CD44, but not c-kit, Lin, CD34, CD45, CD11b, and CD31, compatible with that of mesenchymal stem cells from bone marrow. In coculture conditions with ADSC and human aortic endothelial cells (ECs) under treatment with growth factors, ADSC significantly increased EC viability, migration and tube formation mainly through secretion of vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF). At 4 weeks after transplantation of ADSC into the ischemic mouse hindlimb, the angiogenic scores were improved in the ADSC-treated group, which were evaluated with blood flow by laser Doppler imaging (LDI) and capillary density by immunostaining with anti-CD31 antibody. However, injected ADSC did not correspond to CD31, von Willebrand factor, and alpha-smooth muscle actin-positive cells in ischemic tissue. CONCLUSIONS: These adipose tissue-derived cells demonstrated potential as angiogenic cell therapy for ischemic disease, which appears to be mainly achieved by their ability to secrete angiogenic growth factors.
Subject(s)
Adipose Tissue/cytology , Growth Substances/metabolism , Ischemia/therapy , Stromal Cells/metabolism , Stromal Cells/transplantation , Angiopoietin-1/metabolism , Animals , Aorta/cytology , Cell Division , Cell Movement , Cell Survival , Cells, Cultured , Cytokines/metabolism , Disease Models, Animal , Endothelium, Vascular/cytology , Hepatocyte Growth Factor/metabolism , Hindlimb/blood supply , Humans , Ischemia/pathology , Ischemia/physiopathology , Mice , Mice, Inbred C57BL , Muscle, Smooth, Vascular/cytology , Neovascularization, Physiologic/physiology , Placenta Growth Factor , Pregnancy Proteins/metabolism , Stromal Cells/cytology , Transforming Growth Factor beta/metabolism , Vascular Endothelial Growth Factor A/metabolismABSTRACT
OBJECTIVE: This study investigated post-traumatic stress symptoms in relation to the population affected by the Fukushima Nuclear Disaster, one year after the disaster. Additionally, we investigated social factors, such as forced displacement, which we hypothesize contributed to the high prevalence of post-traumatic stress. Finally, we report of written narratives that were collected from the impacted population. DESIGN AND SETTINGS: Using the Impact of Event Scale-Revised (IES-R), questionnaires were sent to 2,011 households of those displaced from Fukushima prefecture living temporarily in Saitama prefecture. Of the 490 replies; 350 met the criteria for inclusion in the study. Multiple logistic regression analysis was performed to examine several characteristics and variables of social factors as predictors of probable post-traumatic stress disorder, PTSD. RESULTS: The mean score of IES-R was 36.15±21.55, with 59.4% having scores of 30 or higher, thus indicating a probable PTSD. No significant differences in percentages of high-risk subjects were found among sex, age, evacuation area, housing damages, tsunami affected, family split-up, and acquaintance support. By the result of multiple logistic regression analysis, the significant predictors of probable PTSD were chronic physical diseases (OR = 1.97), chronic mental diseases (OR = 6.25), worries about livelihood (OR = 2.27), lost jobs (OR = 1.71), lost social ties (OR = 2.27), and concerns about compensation (OR = 3.74). CONCLUSION: Although there are limitations in assuming a diagnosis of PTSD based on self-report IES-R, our findings indicate that there was a high-risk of PTSD strongly related to the nuclear disaster and its consequent evacuation and displacement. Therefore, recovery efforts must focus not only on medical and psychological treatment alone, but also on social and economic issues related to the displacement, as well.