RESUMO
Controlling the balance of endothelial cells (ECs) and smooth muscle cells (SMCs) in blood vessels is critically important to minimize the risk associated with vascular implants. Extracellular matrix (ECM) plays a key role in controlling the cellular balance, suggesting a promising source of cell-selective peptides. To obtain EC- or SMC-selective peptides, we start by highlighting sequence differences found among ECM molecules as enriched targets for cell-selective peptides. We explored the EC- or SMC-selective performance of tripeptides that are specifically enriched only in collagen type IV, but not in types I, II, III, and V. Collagen type IV was chosen since it is the major ECM in the basement membrane of blood vessels, which separates ECs and SMCs. Among 114 collagen type IV-derived tripeptides pre-screened from in silico analysis, 22 peptides (19%) were found to promote cell-selective adhesion, as determined by peptide array. One of the best performing EC-selective peptides (Cys-Ala-Gly (CAG)) was mixed into an electrospun fine-fiber, a vascular graft material, for practical application. Compared to unmodified fiber, the CAG containing fiber surface was found to enhance adhesion of ECs (+190%) while limiting SMCs (-20%). These results are not only consistent with the hypothesis of ECM as a source of cell selective peptides, but also suggest a new genre of EC- or SMC-selective peptides for tissue engineering applications. Collectively, these findings favorably support the screening approach used to discover new peptides for these purposes.
Assuntos
Prótese Vascular , Colágeno Tipo IV/química , Células Endoteliais/citologia , Músculo Liso Vascular/citologia , Oligopeptídeos/química , Aorta/citologia , Adesão Celular , Linhagem Celular , Matriz Extracelular/química , HumanosRESUMO
The concept of tissue engineered small-caliber vascular grafts (TE-SCVGs) is theoretically ideal. In this study, we evaluated the long-term (more than 1 year) course of TE-SCVGs using a rat carotid arterial replacement model. We fabricated a TE-SCVG scaffold (0.7 mm in diameter) with electrospun nano-scale fibers. Poly-ε-caprolactone was used as a biodegradable polymer. These artificial vessels were then used in carotid arterial replacement performed on Sprague-Dawley rats. The implanted grafts were removed at an early phase (1, 2, 6 weeks), middle phase (12, 24 weeks), and late phase (48, 72 weeks) after implantation. Twenty-nine patent grafts from among the 40 implanted grafts (patency 72.5 %) could be evaluated. No aneurysm formation was observed during the follow-up period. Endothelial cells positive for immunostaining with von Willebrand factor were found to be already attached to the inner surface of the TE-SCVGs in the early phase. The percentage of smooth muscle cell specific marker (α-smooth muscle actin and calponin with fluorescent immunostaining) positive cells, which seemed to be mesenchymal cells in the graft wall, increased with time, while, in contrast, the scaffold material decreased. Even after 72 weeks, however, although the scaffold material had degraded, it had not disappeared completely. These results show that the novel TE-SCVGs we developed were still functioning in the rat carotid arterial circulation after more than 1 year. However, further investigations will be required with regard to regeneration of the SMC layer and the complete degradation of graft materials.
Assuntos
Implante de Prótese Vascular , Prótese Vascular , Artérias Carótidas , Engenharia Tecidual , Implantes Absorvíveis , Animais , Artérias Carótidas/cirurgia , Endotélio Vascular/crescimento & desenvolvimento , Masculino , Células-Tronco Mesenquimais/citologia , Poliésteres , Ratos , Ratos Sprague-Dawley , Regeneração , Alicerces Teciduais , Grau de Desobstrução VascularRESUMO
PURPOSE: The purpose of this study was to evaluate an application of poly(d,l-lactide-co-glycolide) (PLG) scaffold created by electrospinning in a rabbit rotator cuff defect model. METHODS: Forty-two Japanese white rabbits were used in this study. Defects of the infraspinatus tendon were created, and the PLG scaffolds were implanted. Contralateral infraspinatus tendons were reattached without creating defects. Histologic analyses were performed 4, 8, and 16 weeks after the operation, and mechanical evaluations were performed 0, 4, 8, and 16 weeks after the operation. RESULTS: Scaffold fibers remained without dissolution and spindle-shaped cells were observed inside of the scaffold at 4 weeks postoperatively. At 8 weeks, the PLG scaffold had dissolved and bone formation was observed at the scaffold-bone interface. At 16 weeks, the scaffold-bone interface matured and expression of type II collagen was observed. A statistical difference in ultimate failure load was not seen between the scaffold group and reattachment group or normal tendon after 8 weeks postoperatively. The stiffness in the scaffold group was not significantly different from that in the reattachment group at each time point. However, it was significantly weaker than normal tendon at each time point. CONCLUSIONS: Transplantation of cell-free PLG scaffold showed cell migration and type II collagen and proteoglycan expression at the scaffold-bone junction by 16 weeks postoperatively with a sufficient ultimate failure load in a rabbit rotator cuff defect model. CLINICAL RELEVANCE: The PLG scaffold could be applied to bridge rotator cuff defects. The results showed that bridging with scaffold can be equivalent to reattachment.
Assuntos
Ácido Láctico/uso terapêutico , Ácido Poliglicólico/uso terapêutico , Regeneração/fisiologia , Manguito Rotador/fisiologia , Alicerces Teciduais , Animais , Movimento Celular , Colágeno Tipo II/metabolismo , Feminino , Osteogênese/fisiologia , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Proteoglicanas/metabolismo , Coelhos , Reimplante/métodos , Ruptura/patologia , Ruptura/cirurgia , Fatores de TempoRESUMO
We report two cases of inflammatory pseudotumor of the spleen treated by laparoscopic splenectomy. The first patient was a 61-year-old woman with a 3cm splenic tumor detected incidentally by ultrasonography. Computed tomography showed a well-demarcated splenic mass. She underwent hand-assisted laparoscopic splenectomy. The second patient was a 51-year-old man in whom a splenic tumor was found on follow-up computed tomography after resection of occipital malignant neurinoma. Ultrasound and magnetic resonance imaging confirmed a splenic tumor, which showed no uptake on 18F-fluorodeoxyglucose positron emission tomography. Laparoscopic splenectomy was performed. The histopathological diagnosis was inflammatory pseudotumor in both cases. Their postoperative course was uneventful, with a postoperative hospital stay of 11 and 8 days, respectively. Splenectomy is usually performed in patients with splenic tumors because imaging techniques cannot exclude malignancy. Laparoscopic splenectomy may be a useful option for patients with splenic tumors.
Assuntos
Granuloma de Células Plasmáticas/cirurgia , Laparoscopia/métodos , Esplenectomia/métodos , Esplenopatias/cirurgia , Feminino , Granuloma de Células Plasmáticas/diagnóstico , Humanos , Masculino , Pessoa de Meia-Idade , Esplenopatias/diagnósticoRESUMO
BACKGROUND: Bone marrow-derived mesenchymal stem cells (MSCs) can differentiate into various types of cell, and the extracellular matrix (ECM) is acknowledged to be important for the regulation of cell functions. In this study, we demonstrated the effects of ECMs on the differentiation of human bone marrow-derived MSCs into a smooth muscle cell (SMC) lineage. METHODS: Human MSCs (hMSCs) were cultured on dishes coated with 3 types of ECM including laminin (LM), collagen type IV (Col-IV) and fibronectin for 7 days, and simultaneously cultured on a noncoated dish as a control. Cell numbers of these cultured hMSCs were counted, and their expression of SMC-specific genes and proteins was evaluated. hMSCs were then seeded on LM-coated biodegradable sheets and implanted into rat subcutaneous space. After 2 weeks of implantation, these tissues were evaluated. RESULTS: The number of hMSCs was significantly increased by culture on Col-IV-coated dishes. The expression of SMC-specific genes and proteins (alpha-smooth muscle actin, ASMA; h1-calponin, CALP) in hMSC was significantly upregulated from culture on LM-coated dishes. LM-coated sheets showed a significantly increased expression of ASMA and CALP protein in vivo. Moreover, a fully differentiated marker (SM2) was expressed in the in vivo implanted hMSCs in the course of 2 weeks on the LM-coated sheet. CONCLUSION: These results suggest that the signal transduction of the cell-matrix interaction for the differentiation of hMSCs into SMCs was activated when cultured with LM. LM-coated materials may thus be useful for cardiovascular tissue engineering.
Assuntos
Medula Óssea/metabolismo , Matriz Extracelular/metabolismo , Células-Tronco Mesenquimais/citologia , Músculo Liso Vascular/citologia , Miócitos de Músculo Liso/citologia , Engenharia Tecidual , Animais , Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Sistema Cardiovascular/citologia , Diferenciação Celular , Linhagem da Célula , Proliferação de Células , Humanos , Laminina/metabolismo , Células-Tronco Mesenquimais/metabolismo , Miócitos de Músculo Liso/metabolismo , RatosRESUMO
PURPOSE: The purpose of our study was to explore the possibility that an electrospun bioabsorbable scaffold could be used in the treatment of a full-thickness articular defect without the addition of exogenous cells in a rabbit model. METHODS: Two types of poly(D,L-lactide-co-glycolide) (PLG) scaffolds, a solid cylindrical type and a cannulated tubular type, were made with the electrospinning method. Osteochondral defects, 5 mm in diameter and 5 mm in depth, made on the femoral condyles of rabbits were filled with these scaffolds, and the repair process was investigated histologically. RESULTS: In the groups in which the defect was filled with the scaffold, fibrous tissue at the articular surface of the scaffold was observed at postoperative week 2. Thereafter cartilage at the articular surface and bone at the subchondral zone were regenerated, and the repaired cartilage was maintained through postoperative week 24. By contrast, the untreated defect was filled with hematoma at postoperative week 2; thereafter regenerated cartilage and bone were observed. However, the surface of the articular cartilage was not regular, and regenerated cartilage was not well organized. The histologic scores of the groups in which the defect was filled with cannulated tubular electrospun PLG scaffolds were significantly higher than those of the untreated defect group at postoperative weeks 12 and 24 (P < .01). CONCLUSIONS: The electrospun PLG scaffold could repair a 5-mm osteochondral defect created in the rabbit model without exogenous cultured cells. CLINICAL RELEVANCE: The electrospun PLG scaffold could repair full-thickness osteochondral defects. The cannulated type of PLG scaffold has the possibility to lead not only to good regeneration of cartilage but also to easy transplantation by use of a guidewire through the cannulas in the scaffold.
Assuntos
Implantes Absorvíveis , Cartilagem Articular/cirurgia , Fêmur/cirurgia , Alicerces Teciduais/química , Animais , Cartilagem Articular/patologia , Modelos Animais de Doenças , Eletroquímica , Ácido Láctico , Ácido Poliglicólico , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Coelhos , Técnicas de SuturaRESUMO
The blood cells of ascidians accumulate extremely high levels of the transition metal vanadium. We previously isolated four vanadium-binding proteins (Vanabins 1-4) and a homologous protein (VanabinP) from the vanadium-rich ascidian Ascidia sydneiensis samea. In the present study, we identified cDNAs encoding five different Vanabin2-related proteins in A. sydneiensis samea blood cells. It was notable that the sequences of the encoded proteins vary from that of Vanabin2 at up to 14 specific positions, while both the polypeptide length and the 18 cysteine residues were completely conserved. The most divergent protein, named 14MT, differed from Vanabin2 at all 14 positions. Using immobilized metal-ion affinity chromatography, we found that Vanabin2 and 14MT have the same metal-ion selectivity, but the overall affinity of 14MT for VO(2+) is higher than that of Vanabin2. Binding number for VO(2+) ions was the same between Vanabin2 and 14MT as assessed by gel filtration. These results suggested that sequence variations were under strict evolutionary constraints and high-affinity binding sites for VO(2+) are conserved among Vanabin2 variants.
Assuntos
Proteínas Sanguíneas/genética , Urocordados/genética , Vanádio/metabolismo , Sequência de Aminoácidos , Animais , Células Sanguíneas/metabolismo , Proteínas Sanguíneas/metabolismo , Sequência Conservada , Cisteína/química , DNA Complementar/genética , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Ligação Proteica , Homologia de Sequência de Aminoácidos , Urocordados/metabolismo , Vanádio/químicaRESUMO
Pancreatic ductal adenocarcinomas are thought to arise from noninvasive, intraductal precursor lesions called pancreatic intraepithelial neoplasias (PanIN). The study of PanINs holds great promise for the identification of early detection markers and effective cancer-preventing strategies. Cyclooxygenase-2 (COX-2) represents an intriguing target for therapeutic and preventive approaches in various human malignancies. The aim of the present study was to evaluate the efficacy of a selective COX-2 inhibitor to prevent the progression of PanINs in a conditional Kras(G12D) mouse model. Offspring of LSL-KRAS(G12D) x PDX-1-Cre intercrosses were randomly allocated to a diet supplemented with the selective COX-2 inhibitor nimesulide (400 ppm) or a control diet. After 10 months, animals were sacrificed. Successful recombination in the pancreas was evaluated by PCR. The pancreas of KRAS(G12D);PDX-1-Cre mice was analyzed for the presence of murine PanINs. Animals fed the COX-2 inhibitor had significantly fewer PanIN-2 and PanIN-3 lesions than control animals (P < 0.05). Ten percent of all pancreatic ducts in the nimesulide-fed animals showed PanIN-2 or PanIN-3 lesions, whereas 40% of the pancreatic ducts in the control animals had PanIN-2 or PanIN-3 lesions. Intrapancreatic prostaglandin E(2) levels were reduced in nimesulide-fed animals. Immunohistochemistry confirmed COX-2 expression in early and late PanINs. In summary, we found that the selective COX-2 inhibitor nimesulide delays the progression of pancreatic cancer precursor lesions in a preclinical animal model. These data highlight the importance of COX-2 in the development of pancreatic cancer. Inhibition of COX-2 may represent an intriguing strategy to prevent pancreatic cancer in high-risk patients.
Assuntos
Carcinoma Ductal Pancreático/prevenção & controle , Inibidores de Ciclo-Oxigenase 2/uso terapêutico , Modelos Animais de Doenças , Genes ras/fisiologia , Neoplasias Pancreáticas/prevenção & controle , Sulfonamidas/uso terapêutico , Animais , Carcinoma Ductal Pancreático/enzimologia , Carcinoma Ductal Pancreático/patologia , Dinoprostona/metabolismo , Progressão da Doença , Marcação de Genes , Genes ras/genética , Humanos , Técnicas Imunoenzimáticas , Integrases/metabolismo , Camundongos , Mutação/genética , Neoplasias Pancreáticas/enzimologia , Neoplasias Pancreáticas/patologiaRESUMO
PURPOSE: This study was undertaken to investigate whether thioltransferase (TTase) exhibits dehydroascorbate (DHA) reductase activity in human lens epithelial cells. METHODS: TTase was investigated for DHA reductase activity in vitro by the method of glutathione reductase-coupled spectrophotometric assay. DHA reductase activities of human lens epithelial (HLE-B3) cell lysate and TTase-depleted HLE-B3 cell lysate were determined with a 6-deoxy-6-fluoro-DHA probe and 19F-nuclear magnetic resonance (NMR) spectroscopy. TTase-overexpressing and -depleted HLE-B3 cells were investigated for DHA reductase activity. RESULTS: TTase showed DHA reductase activity at a Km of 0.15 mM and Vmax of 35 nmol/min. Investigation of the DHA reductase activity in human lens epithelial (HLE-B3) cell lysate, by using a 6-deoxy-6-fluoro-DHA probe and 19F-NMR spectroscopy, revealed that cell lysate possesses significant DHA reductase activity. This activity decreased extensively when TTase was depleted from the cell lysate by immunoprecipitation. In a cell-free system with externally added DHA, nearly 70% of the recycling ability was diminished when TTase was removed from the lysate. The TTase-overexpressing cells increased DHA reductase activity twofold. HLE-B3 cells showed an ability to take up and recycle DHA, and this ability was increased approximately twofold in the TTase-transfected cells. Suppression of TTase in HLE-B3 cells by an antisense cDNA strategy resulted in a 77% decrease in DHA reductase activity. CONCLUSIONS: The data provide evidence that TTase plays a major role in ascorbic acid recycling in human lens epithelial cells.
Assuntos
Ácido Ascórbico/metabolismo , Células Epiteliais/enzimologia , Cristalino/enzimologia , Oxirredutases/metabolismo , Oxirredutases/fisiologia , Proteína Dissulfeto Redutase (Glutationa) , Anticorpos Bloqueadores/farmacologia , Linhagem Celular , Células Epiteliais/efeitos dos fármacos , Corantes Fluorescentes , Glutarredoxinas , Glutationa/farmacologia , Humanos , Cristalino/citologia , Espectroscopia de Ressonância Magnética , Oxirredutases/antagonistas & inibidores , Fatores de TempoRESUMO
PURPOSE: Metabolomics, or metabolic profiling, is an emerging discipline geared to providing information on a large number of metabolites, as a complement to genomics and proteomics. In the current study, a fluorine-labeled derivative of ascorbic acid (F-ASA), a major antioxidant- and UV-trapping molecule in the aqueous humor and the lens, was used to investigate the extent to which the lens accumulates potentially toxic degradation products of vitamin C. METHODS: Human lens epithelial cells (HLE-B3) and rat lenses were exposed to hyperglycemic or oxidative stress in vitro or in vivo and probed for accumulation of F-ASA, fluoro-dehydroascorbate (F-DHA), fluoro-2,3-diketogulonate (F-DKG), and their degradation products in protein-free extracts, by proton-decoupled 750-MHz (19)F-nuclear magnetic resonance (NMR) spectroscopy. RESULTS: F-ASA and F-DHA were taken up into HLE B-3 cells by an Na(+)-dependent transporter. Their uptake was unexpectedly only slightly affected by hyperglycemia in vitro, unless glutathione was severely depleted. Glycemic stress catalyzed oxidation of F-ASA into a single novel F-compound at -212.4 ppm, whereas F-DHA and F-DKG were the major degradation products observed after GSH depletion. In contrast, F-ASA uptake was markedly suppressed in diabetic cataractous rat lenses, which accumulated both the F-DHA and the -212.4-ppm compound. In an unexpected finding, the latter formed only from F-ASA and not F-DHA or F-DKG, suggesting a novel pathway of in vivo F-ASA degradation. Both the cells and the intact rat and human lenses were permeable to several advanced F-ASA and F-DHA degradation products, except F-DKG. The unknown compound at -212.4 ppm was the only F-ASA degradation product that spontaneously formed in rabbit aqueous humor upon incubation with F-ASA. CONCLUSIONS: These studies suggest the existence of a novel ascorbic-acid-degradation pathway in the lens and aqueous humor that is influenced by the nature of the oxidant stress. Under similar culture conditions, intact lenses are more prone to hyperglycemia-mediated oxidant stress than are lens epithelial cells, but both are permeable to various F-ASA degradation products, the structure and biological roles of which remain to be established.
Assuntos
Ácido Ascórbico/análogos & derivados , Ácido Ascórbico/metabolismo , Células Epiteliais/metabolismo , Cristalino/metabolismo , Espectroscopia de Ressonância Magnética , Ácido 2,3-Dicetogulônico/metabolismo , Adulto , Idoso , Animais , Transporte Biológico , Butionina Sulfoximina/farmacologia , Catarata/induzido quimicamente , Catarata/metabolismo , Técnicas de Cultura de Células , Citocalasina B/farmacologia , Ácido Desidroascórbico/metabolismo , Inibidores Enzimáticos/farmacologia , Radioisótopos de Flúor , Galactose/farmacologia , Glucose/farmacologia , Glutationa/antagonistas & inibidores , Glutationa/metabolismo , Humanos , Hiperglicemia/metabolismo , Masculino , Pessoa de Meia-Idade , Técnicas de Cultura de Órgãos , Estresse Oxidativo , Coelhos , Ratos , Ratos Sprague-DawleyRESUMO
Metabolomic mapping is an emerging discipline geared at providing information on a large number of metabolites as a complement to genomics and proteomics. Here we have probed ascorbic acid homeostasis and degradation in diabetes using 6-deoxy-6-fluoro ascorbic acid (F-ASA) and 750 MHz (19)F-nuclear magnetic resonance (NMR) spectroscopy with proton decoupling In vitro, Cu(2+)-mediated degradation of F-ASA revealed the formation of 4 major stable degradation products at 24 hours. However, when normal or diabetics rats were injected with F-ASA intraperitoneally (IP) for 4 days, up to 20 fluorine-labeled compounds were observed in the urine. Their composition resembled, in part, metal catalyzed degradation of F-ASA and was not explained by spontaneous degradation in the urine. Diabetes led to a dramatic increase in urinary F-ASA loss and a relative decrease in most other urinary F-compounds. Diabetes tilted F-ASA homeostasis toward oxidation in liver (P <.01), kidney (P <.01), spleen (P <.01), and plasma (P <.01), but tended to decrease oxidation in brain, adrenal glands, and heart. Surprisingly, however, besides the major oxidation product fluoro-dehydroascorbic acid (F-DHA), no F-ASA advanced catabolites were detected in tissues at 5 micromol/L sensitivity. These findings not only confirm the key role of the kidney in diabetes-mediated loss of ascorbic acid, but demonstrate that only selected tissues are prone to increased oxidation in diabetes. While the structure of most degradation products needs to be established, the method illustrates the power of high resolution (19)F-NMR spectroscopy for the mapping of complex metabolomic pathways in disease states.
Assuntos
Ácido Ascórbico/análogos & derivados , Ácido Ascórbico/metabolismo , Diabetes Mellitus Experimental/metabolismo , Ácido 2,3-Dicetogulônico/síntese química , Animais , Ácido Ascórbico/sangue , Ácido Ascórbico/urina , Peso Corporal , Ácido Desidroascórbico/sangue , Ácido Desidroascórbico/urina , Flúor , Radioisótopos de Flúor , Homeostase , Cinética , Espectroscopia de Ressonância Magnética , Masculino , Tamanho do Órgão , Concentração Osmolar , Oxirredução , Ratos , Ratos Sprague-DawleyRESUMO
Mesenchymal stem cells (MSC) have been used recently for the treatment of autoimmune diseases in murine animal models due to the immunoregulatory capacity. Current utilization of MSC requires cells in certain quantity with multiple courses of administration, leading to limitation in clinical usage. Here we efficiently treated collagen-induced arthritis rats with a single local implantation with reduced number of MSC (2â¼20% of previous studies) with nano-fiber poly-lactic-co-glycolic acid (nano-fiber) scaffold. MSC seeded on nano-fiber scaffold suppressed arthritis and bone destruction due to inhibition of systemic inflammatory reaction and immune response by suppressing T cell proliferation and reducing anti- type II collagen antibody production. In vivo tracing of MSC demonstrated that these cells remained within the scaffold without migrating to other organs. Meanwhile, in vitro culture of MSC with nano-fiber scaffold significantly increased TGF-ß1 production. These results indicate an efficient utilization of MSC with the scaffold for destructive joints in rheumatoid arthritis by a single and local inoculation. Thus, our data may serve as a new strategy for MSC-based therapy in inflammatory diseases and an alternative delivery method for bone destruction treatment.
Assuntos
Artrite Experimental/terapia , Transplante de Células-Tronco Mesenquimais/métodos , Células-Tronco Mesenquimais/fisiologia , Adulto , Animais , Artrite Experimental/imunologia , Linfócitos T CD4-Positivos/fisiologia , Proliferação de Células , Citocinas/metabolismo , Feminino , Humanos , Ácido Láctico/química , Nanofibras/química , Poliésteres , Ácido Poliglicólico/química , Polímeros/química , Ratos Endogâmicos Lew , Alicerces Teciduais/química , Fator de Crescimento Transformador beta1/metabolismo , Adulto JovemRESUMO
OBJECTIVES: Arterial graft spasm occasionally causes circulatory collapse immediately following coronary artery bypass graft. The aim of this study is to evaluate the efficacy of our developed materials, which were composed of milrinone (phosphodiesterase III inhibitor) or diltiazem (calcium-channel blocker), with nano-scaled fibre made of biodegradable polymer to prevent arterial spasm. METHODS: Milrinone- or diltiazem-releasing biodegradable nano-scaled fibres were fabricated by an electrospinning procedure. In vivo milrinone- or diltiazem-releasing tests were performed to confirm the sustained release of the drugs. An in vivo arterial spasm model was established by subcutaneous injection of noradrenalin around the rat femoral artery. Rats were randomly divided into four groups as follows: those that received 5 mg of milrinone-releasing biodegradable nano-scaled fibre (group M, n = 14); 5 mg of diltiazem-releasing biodegradable nano-scaled fibre (group D, n = 12); or those that received fibre without drugs (as a control; group C, n = 14) implanted into the rat femoral artery. In the fourth group, sham operation was performed (group S, n = 10). One day after the implantation, noradrenalin was injected in all groups. The femoral arterial blood flow was measured continuously before and after noradrenalin injection. The maximum blood flow before noradrenalin injection and minimum blood flow after noradrenalin injection were measured. RESULTS: In vivo drug-releasing test revealed that milrinone-releasing biodegradable nano-scaled fibre released 78% of milrinone and diltiazem-releasing biodegradable nano-scaled fibre released 50% diltiazem on the first day. The ratios of rat femoral artery blood flow after/before noradrenalin injection in groups M (0.74 ± 0.16) and D (0.72 ± 0.05) were significantly higher than those of groups C (0.54 ± 0.09) and S (0.55 ± 0.16) (P < 0.05). CONCLUSION: Noradrenalin-induced rat femoral artery spasm was inhibited by the implantation of milrinone-releasing biodegradable nano-scaled fibre or diltiazem-releasing biodegradable nano-scaled fibre. These results suggested that our materials might be effective for the prevention of arterial graft spasm after coronary artery bypass graft.
Assuntos
Implantes Absorvíveis , Arteriopatias Oclusivas/prevenção & controle , Diltiazem/administração & dosagem , Portadores de Fármacos , Artéria Femoral/efeitos dos fármacos , Milrinona/administração & dosagem , Nanofibras , Vasoconstrição/efeitos dos fármacos , Vasodilatadores/administração & dosagem , Animais , Arteriopatias Oclusivas/induzido quimicamente , Arteriopatias Oclusivas/fisiopatologia , Bloqueadores dos Canais de Cálcio/administração & dosagem , Constrição Patológica , Modelos Animais de Doenças , Artéria Femoral/fisiopatologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Norepinefrina , Inibidores da Fosfodiesterase 3/administração & dosagem , Poliglactina 910 , Ratos , Ratos Sprague-Dawley , Fluxo Sanguíneo Regional , Fatores de TempoRESUMO
BACKGROUND: Both rapid endothelialization and the prevention of intimal hyperplasia are essential to improve the patency of small-caliber vascular grafts (SCVGs). Using the peptide array based screening system, we identified the peptide CAG (cysteine-alanine-glycine), which has a high affinity for endothelial cells and a low adhesive property for smooth muscle cells (SMCs). In this article, we report an in vivo analysis of novel vascular grafts that were constructed with a biodegradable polymer (poly-ε-caprolactone [PCL]) containing CAG peptide. METHODS: The novel SCVG, which measured 0.7 mm in diameter and 7 mm in length, was fabricated using the electrospinning technique. Carotid arterial replacement was performed on Sprague-Dawley rats using SCVGs with (group CAG) or without CAG (group C). Histologic and biochemical assessments were performed at 1, 2, and 6 weeks after implantation. RESULTS: The ratio of endothelialization was significantly higher in group CAG compared with group C (CAG versus C, 64.4±20.0% versus 42.1±8.9% at 1 week; p=0.017; 98.2±2.3% versus 72.7±12.9% at 2 weeks; p=0.001; and 97.4±4.6% versus 76.7±5.4% at 6 weeks; p<0.001). Additionally, Western blot analysis showed that the level of endothelial nitric oxide synthase (eNOS) at 1 week in group CAG was significantly higher than that in group C (CAG versus C, 1.20±0.37 versus 0.34±0.16; p=0.013), and that α-smooth muscle actin (ASMA) at 6 weeks in group CAG was significantly lower than that in group C (CAG versus C, 0.89±0.06 versus 1.25±0.22; p=0.04). CONCLUSIONS: The graft with CAG promoted rapid endothelialization and the potential for inhibition of intimal hyperplasia.
Assuntos
Arteriopatias Oclusivas/cirurgia , Prótese Vascular , Caproatos , Endotélio Vascular/patologia , Lactonas , Músculo Liso Vascular/patologia , Implantes Absorvíveis , Animais , Modelos Animais de Doenças , Hiperplasia , Masculino , Miniaturização , Desenho de Prótese , Ratos , Ratos Sprague-DawleyRESUMO
Tissue engineering is an effective approach for the treatment of bone defects. Statins have been demonstrated to promote osteoblastic differentiation of bone marrow-derived stromal cells (BMSCs). Electrospun biodegradable fibers have also shown applicability to drug delivery in the form of bone tissue engineered scaffolds with nano- to microscale topography and high porosity similar to the natural extracellular matrix (ECM). The aim of this study was to investigate the feasibility of a simvastatin-releasing, biodegradable, nano- to microscale fiber scaffold (SRBFS) for bone tissue engineering with BMSCs. Simvastatin was released from SRBFS slowly. BMSCs were observed to spread actively and rigidly adhere to SRBFS. BMSCs on SRBFS showed an increase in alkaline phosphatase activity 2 weeks after cell culture. Furthermore, osteoclastogenesis was suppressed by SRBFS in vitro. The new bone formation and mineralization in the SRBFS group were significantly better than in the biodegradable fiber scaffold (BFS) without simvastatin 12 weeks after implantation of the cell-scaffold construct into an ectopic site on the murine back. These results suggest that SRBFS promoted osteoblastic differentiation of BMSCs in vitro and in vivo, and demonstrate feasibility as a bone engineering scaffold.
Assuntos
Implantes Absorvíveis , Desenvolvimento Ósseo/fisiologia , Transplante de Células-Tronco Mesenquimais/instrumentação , Células-Tronco Mesenquimais/fisiologia , Osteogênese/fisiologia , Sinvastatina/administração & dosagem , Alicerces Teciduais , Animais , Desenvolvimento Ósseo/efeitos dos fármacos , Células Cultivadas , Implantes de Medicamento/administração & dosagem , Implantes de Medicamento/química , Estudos de Viabilidade , Células-Tronco Mesenquimais/efeitos dos fármacos , Camundongos , Osteogênese/efeitos dos fármacos , Desenho de Prótese , Sinvastatina/química , Engenharia TecidualRESUMO
Pulmonary venous obstruction (PVO) after correction of total anomalous pulmonary venous connection (TAPVC) frequently occurs due to intimal-hyperplasia and the required re-operation. We have developed a novel sustained-release drug delivery system, using Tacrolimus-eluting biodegradable nano-fiber (TEBN). It consists of nano-scale fiber composed of biodegradable polymer and Tacrolimus. This study evaluated the effects of TEBN for prevention of venous anastomotic stricture in a rat model to apply to PVO operation. Tacrolimus was incorporated into poly (L-lactide-co-glycolide). The venous stricture model was made by rat inferior vena cava anastomosis. The IVC anastomosis was covered with TEBN with 1.0 wt% Tacrolimus (n=12) or without TEBN as a control (n=12), and evaluated histologically at 1, 2, and 4 weeks after operation. The ratio of intimal area was significantly reduced in the TEBN group compared with the control group (ratio; 1 week: 0.43+/-0.26 vs. 0.07+/-0.04, P=0.04, 2 weeks: 0.39+/-0.19 vs. 0.05+/-0.02, P=0.01, 4 weeks: 0.31+/-0.15 vs. 0.09+/-0.04, P=0.03, control vs. TEBN, respectively). Histological findings showed endothelialization along the inner surface of the vein even in TEBN. The TEBN reduced intimal hyperplasia and preserved endothelialization even in a venous stricture. These results suggested that this strategy might be useful for prevention of recurrent PVO after TAPVC correction.
Assuntos
Implantes Absorvíveis , Fármacos Cardiovasculares/administração & dosagem , Stents Farmacológicos , Nanoestruturas , Poliglactina 910 , Pneumopatia Veno-Oclusiva/prevenção & controle , Tacrolimo/administração & dosagem , Veia Cava Inferior/efeitos dos fármacos , Anastomose Cirúrgica , Animais , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Humanos , Hiperplasia , Masculino , Teste de Materiais , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/patologia , Desenho de Prótese , Pneumopatia Veno-Oclusiva/etiologia , Pneumopatia Veno-Oclusiva/patologia , Ratos , Ratos Wistar , Fatores de Tempo , Túnica Íntima/efeitos dos fármacos , Túnica Íntima/patologia , Veia Cava Inferior/patologia , Veia Cava Inferior/cirurgiaRESUMO
OBJECTIVE: We developed a novel sustained drug-eluting device using tacrolimus-eluting biodegradable nanofiber to prevent anastomotic stricture and evaluated the effects in a rat abdominal aortic anastomosis model. METHODS: In vitro and in vivo tacrolimus release tests for tacrolimus-eluting biodegradable nanofiber were performed to confirm its sustained release. To verify the prevention of anastomotic stricture, tacrolimus-eluting biodegradable nanofiber was placed around the end-to-end anastomosis of abdominal aorta in rats. Five rats were allocated to the following 5 groups: (1) control without tacrolimus-eluting biodegradable nanofiber, (2) 5 mg of nanofiber only (0 wt% of tacrolimus), (3) 5 mg of tacrolimus-eluting biodegradable nanofiber containing 0.04 wt% of tacrolimus, (4) 5 mg of tacrolimus-eluting biodegradable nanofiber containing 0.1 wt% of tacrolimus, and (5) 5 mg of tacrolimus-eluting biodegradable nanofiber containing 1.0 wt% of tacrolimus. Morphometric and histologic analyses including immunohistochemistry were performed in each of the groups 2 weeks after the operation. RESULTS: The tacrolimus-eluting biodegradable nanofiber gradually released tacrolimus for at least 1 month in vitro and in vivo. The ratio of intimal area was significantly reduced in the 1.0 wt% tacrolimus-eluting biodegradable nanofiber group compared with the other groups (0.26, 0.24, 0.25, 0.21, and 0.08 in control, 0 wt%, 0.04 wt%, 0.1 wt%, and 1.0 wt%, respectively, P < .05). The cells, which constitute intimal hyperplasia, were positive for smooth muscle actin and SMemb, and factor VIII revealed that endothelial cells covered the surface of the aortic lumen even in the 1.0 wt% tacrolimus-eluting biodegradable nanofiber group in immunohistochemistry. CONCLUSION: Tacrolimus-eluting biodegradable nanofiber reduced neointimal hyperplasia and preserved endothelialization. This device may be useful in the prevention of anastomotic stricture.
Assuntos
Aorta Abdominal/cirurgia , Doenças da Aorta/prevenção & controle , Nanoestruturas , Complicações Pós-Operatórias/prevenção & controle , Tacrolimo/administração & dosagem , Anastomose Cirúrgica , Animais , Materiais Biocompatíveis , Constrição Patológica/prevenção & controle , Modelos Animais , Ratos , Ratos WistarRESUMO
OBJECTIVE: Mucinous cystic neoplasm (MCN) and intraductal papillary mucinous neoplasm of the branch duct type (IPMN-BD) differ in biological and clinical behaviors, but MCN is often misdiagnosed as IPMN-BD. The purpose of this study was to find useful markers for the differential diagnosis of MCN and IPMN-BD. METHODS: Immunohistochemically, the expression of the 2 types of mucin (MUC) 1 (MUC1/DF3 and MUC1/CORE), MUC2, MUC5AC, MUC6, human gastric mucin (HGM), caudal-related homeobox transcription factor 2 (CDX2), CD10, cytokeratin (CK) 7, and CK20 was examined in 7 cases of MCN and 16 cases of IPMN-BD. RESULTS: Expression frequencies in MCN and IPMN-BD were 100% versus 44% for MUC1/DF3, 86% versus 31% for MUC1/CORE, 57% versus 19% for MUC2, 86% versus 100% for MUC5AC, 57% versus 88% for MUC6, 86% versus 100% for HGM, 57% versus 0% for CDX2, 71% versus 0% for CD10, 100% versus 69% for CK7, and 86% versus 6% for CK20. CONCLUSIONS: Mucin 1/DF3, MUC1/CORE, CDX2, CD10, and CK20 were expressed significantly more frequently in MCN than in IPMN-BD. In particular, CD10 and CK20 showed marked differences in immunohistochemical sensitivity and specificity between MCN and IPMN-BD. It is therefore proposed that CD10 and CK20 may be used for the differential diagnosis of MCN and IPMN-BD.
Assuntos
Carcinoma Ductal Pancreático/patologia , Carcinoma Papilar/patologia , Cistadenocarcinoma Mucinoso/patologia , Queratina-20/análise , Neprilisina/análise , Neoplasias Pancreáticas/patologia , Idoso , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Papilar/metabolismo , Cistadenocarcinoma Mucinoso/metabolismo , Diagnóstico Diferencial , Feminino , Humanos , Imuno-Histoquímica , Queratina-7/análise , Masculino , Pessoa de Meia-Idade , Mucina-5AC/análise , Mucina-1/análise , Mucina-2/análise , Mucina-6/análise , Neoplasias Pancreáticas/metabolismoRESUMO
OBJECTIVES: Epidemiologic studies suggest that fish oil, rich in n-3 polyunsaturated fatty acids (PUFA), possesses antitumor activity, whereas n-6 PUFAs may stimulate the development of cancers. The aim of this study was to evaluate the effects of n-6 and n-3 PUFAs on the growth of pancreatic cancer. METHODS: The n-6 PUFA arachidonic acid (AA) stimulated the growth of cyclooxygenase (COX) 2 positive human pancreatic cancer (PaCa) cells, which was mediated by COX-2 generated prostaglandin E2 (PGE2) binding to EP2 and EP4 receptors. In contrast, the n-3 PUFA eicosapentaenoic acid decreased the growth of COX-2-positive and COX-2-negative PaCa cells. The COX-2-dependent mechanism of eicosapentaenoic acid was mediated by binding of PGE3 to EP2 and EP4 receptors. Dietary intake of n-3 PUFAs decreased the growth of pancreatic cancers in a xenograft model, which was accompanied by a decrease of PGE2 and an increase of PGE3 in the tumors. CONCLUSIONS: Our studies provide evidence that n-3 PUFAs possess antitumor activities, whereas n-6 PUFAs stimulate pancreatic tumor growth. The opposite effects of n-3 and n-6 PUFAs are mediated by the formation of different prostaglandin species. n-3 PUFAs may prove beneficial as monotherapy or combination therapy with standard chemotherapeutic agents in pancreatic cancer patients.
Assuntos
Divisão Celular/efeitos dos fármacos , Ácidos Graxos Ômega-3/farmacologia , Ácidos Graxos Ômega-6/farmacologia , Neoplasias Pancreáticas/patologia , Animais , Linhagem Celular Tumoral , AMP Cíclico/metabolismo , Ciclo-Oxigenase 2/metabolismo , Humanos , Camundongos , Modelos Animais , Neoplasias Pancreáticas/embriologia , Reação em Cadeia da Polimerase , Prostaglandina-Endoperóxido Sintases/metabolismo , Prostaglandinas/análise , RNA Neoplásico/genética , RNA Neoplásico/isolamento & purificação , TransfecçãoRESUMO
OBJECTIVES: There is strong evidence for an important role of cyclooxygenase (COX) 2 and COX-2-generated PGE2 during pancreatic tumorigenesis. Cyclooxygenase 2 has therefore become a potential chemotherapeutic target for pancreatic cancer. However, recent studies raised concerns regarding the safety of selective COX-2 inhibitors. Although the benefits of COX-2 inhibition may eventually outweigh the associated cardiovascular risks, there are a number of alternative targets for inhibiting the formation of PGE2 in human tumors that may prove less harmful to the patient. This study aimed at analyzing the expression of various proteins involved in the generation of PGE2 in human pancreatic cancers. METHODS AND RESULTS: Real-time polymerase chain reaction and Western blot analyses demonstrated overexpression of cytoplasmic phospholipase A2, COX-2, cytoplasmic prostaglandin E synthase, and microsomal prostaglandin E synthases 1 and 2 in most human pancreatic cancers when compared with matched normal pancreas. Immunohistochemistry revealed expression of these proteins predominantly by pancreatic cancer cells. Variable expression of these proteins was also confirmed in several human pancreatic cancer cell lines. CONCLUSIONS: Our studies demonstrated for the first time that various proteins involved in the generation of PGE2 are overexpressed in human pancreatic cancers. These proteins may represent potentially novel targets for the therapy of pancreatic cancers.