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1.
Int J Mol Sci ; 22(16)2021 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-34445404

RESUMO

Chronic UV radiation causes oxidative stress and inflammation of skin and blood cells. Therefore, in this study, we assessed the effects of cannabidiol (CBD), a natural phytocannabinoid with antioxidant and anti-inflammatory properties, on the phospholipid (PL) and ceramide (CER) profiles in the plasma of nude rats irradiated with UVA/UVB and treated topically with CBD. The results obtained showed that UVA/UVB radiation increased the levels of phosphatidylcholines, lysophospholipids, and eicosanoids (PGE2, TxB2), while downregulation of sphingomyelins led to an increase in CER[NS] and CER[NDS]. Topical application of CBD to the skin of control rats significantly upregulated plasma ether-linked phosphatidylethanolamines (PEo) and ceramides. However, CBD administered to rats irradiated with UVA/UVB promoted further upregulation of CER and PEo and led to significant downregulation of lysophospholipids. This was accompanied by the anti-inflammatory effect of CBD, manifested by a reduction in the levels of proinflammatory PGE2 and TxB2 and a dramatic increase in the level of anti-inflammatory LPXA4. It can therefore be suggested that topical application of CBD to the skin of rats exposed to UVA/UVB radiation prevents changes in plasma phospholipid profile resulting in a reduction of inflammation by reducing the level of LPE and LPC species and increasing antioxidant capacity due to upregulation of PEo species.


Assuntos
Canabidiol/administração & dosagem , Ceramidas/sangue , Eicosanoides/sangue , Fosfolipídeos/sangue , Raios Ultravioleta/efeitos adversos , Administração Tópica , Animais , Canabidiol/farmacologia , Ceramidas/efeitos da radiação , Cromatografia de Fase Reversa , Eicosanoides/efeitos da radiação , Masculino , Fosfolipídeos/efeitos da radiação , Ratos , Ratos Nus , Espectrometria de Massas em Tandem
2.
Methods Mol Biol ; 2320: 65-73, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34302648

RESUMO

In this chapter, we introduce the method for fabricating thick and anisotropic cardiac tissue for heart regeneration. Aligned and biodegradable nanofiber can be prepared by electrospinning Food and Drug Administration-approved poly (lactic-co-glycolic acid) on a rotating drum. After the nanofibers are transferred on to a polydimethylsiloxane frame, the cardiomyocytes could be plated on the nanofiber to form thick and anisotropic cardiac tissue rapidly. Cardiac tissue-like construct could be easily created by one-step method, and transplanted onto the hearts of myocardium infarction models and lead to their functional recovery.


Assuntos
Infarto do Miocárdio/terapia , Miócitos Cardíacos/citologia , Nanofibras/química , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/química , Animais , Anisotropia , Células Cultivadas , Masculino , Miocárdio/citologia , Ratos , Ratos Nus , Engenharia Tecidual/métodos , Tecidos Suporte/química
3.
Am J Physiol Lung Cell Mol Physiol ; 321(2): L416-L428, 2021 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-34189964

RESUMO

Pulmonary hypertension (PH) is a devastating disease characterized by progressive elevation of pulmonary vascular resistance, right ventricular failure, and ultimately death. We have shown previously that insulin receptor substrate 2 (IRS2), a molecule highly critical to insulin resistance and metabolism, has an anti-inflammatory role in Th2-skewed lung inflammation and pulmonary vascular remodeling. Here, we investigated the hypothesis that IRS2 has an immunomodulatory role in human and experimental PH. Expression analysis showed that IRS2 was significantly decreased in the pulmonary vasculature of patients with pulmonary arterial hypertension and in rat models of PH. In mice, genetic ablation of IRS2 enhanced the hypoxia-induced signaling pathway of Akt and Forkhead box O1 (FOXO1) in the lung tissue and increased pulmonary vascular muscularization, proliferation, and perivascular macrophage recruitment. Furthermore, mice with homozygous IRS2 gene deletion showed a significant gene dosage-dependent increase in pulmonary vascular remodeling and right ventricular hypertrophy in response to hypoxia. Functional studies with bone marrow-derived macrophages isolated from homozygous IRS2 gene-deleted mice showed that hypoxia exposure led to enhancement of the Akt and ERK signaling pathway followed by increases in the pro-PH macrophage activation markers, vascular endothelial growth factor-A and arginase 1. Our data suggest that IRS2 contributes to anti-inflammatory effects by regulating macrophage activation and recruitment, which may limit the vascular inflammation, remodeling, and right ventricular hypertrophy that are seen in PH pathology. Restoring the IRS2 pathway may be an effective therapeutic approach for the treatment of PH and right heart failure.


Assuntos
Hipertensão Pulmonar/metabolismo , Hipóxia/metabolismo , Proteínas Substratos do Receptor de Insulina/metabolismo , Remodelação Vascular , Animais , Modelos Animais de Doenças , Feminino , Proteína Forkhead Box O1/genética , Proteína Forkhead Box O1/metabolismo , Humanos , Hipertensão Pulmonar/genética , Hipertensão Pulmonar/patologia , Hipóxia/genética , Hipóxia/patologia , Inflamação/genética , Inflamação/metabolismo , Inflamação/patologia , Proteínas Substratos do Receptor de Insulina/genética , Masculino , Camundongos , Camundongos Knockout , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Ratos Nus
4.
Biomed Res Int ; 2021: 8875503, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33628824

RESUMO

In order to explore the specific mechanism of YiqiChutan formula (YQCTF) in inhibiting the angiogenesis of lung cancer and its relationship with delta-like ligand 4- (DLL4-) Notch signaling, 30 healthy BALB/c-nu/nu rats were selected and divided into three groups: A549 group (implanted with lung adenocarcinoma cell line A549), NCI-H460 group (implanted with human lung large-cell carcinoma cell line NCI-H460), and NCI-H446 group (implanted with human lung small cell carcinoma cell line NCI-H446) for constructing lung cancer transplanted tumor models. After modeling, the group treated with normal saline was taken as control group, 200 mg/kg of YQCTF was adopted for intervention, and the tumor volume and growth inhibition rate were compared with the vascular targeted inhibitor Sorafenib. HE staining, CD31 fluorescent antibody staining, and microelectron microscopy were adopted to observe the neovascular endothelial cells of the transplanted tumor. The expression of VEGF, HIF-1α, DLL4, and Notch-1 in the transplanted tumors in each group was detected by Western blot and RT-PCR at the protein level or mRNA level. Compared with the control group, the YQCTF-treated group had obvious inhibitory effect on lung cancer transplanted tumor and lung cancer angiogenesis. In the YQCTF-treated group, the density of angiogenesis decreased significantly and the vascular lumen structure also decreased, and the expression levels of VEGF, HIF-1α, DLL4, and Notch-1 in the YQCTF-treated group were all lower than those in the control group. YQCTF could inhibit the growth of lung cancer transplanted tumor through antiangiogenesis, and it could also reduce the amount of angiogenesis in lung cancer transplanted tumor. In addition, the generation of lumen structure was also hindered, which was realized through the VEGF signaling pathway and DLL4-Notch signaling pathway.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Adenocarcinoma de Pulmão , Proteínas de Ligação ao Cálcio/metabolismo , Medicamentos de Ervas Chinesas/farmacologia , Neoplasias Pulmonares , Proteínas de Neoplasias/metabolismo , Neovascularização Patológica , Receptores Notch/metabolismo , Transdução de Sinais/efeitos dos fármacos , Células A549 , Adenocarcinoma de Pulmão/irrigação sanguínea , Adenocarcinoma de Pulmão/tratamento farmacológico , Adenocarcinoma de Pulmão/metabolismo , Adenocarcinoma de Pulmão/patologia , Animais , Humanos , Neoplasias Pulmonares/irrigação sanguínea , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Masculino , Neovascularização Patológica/tratamento farmacológico , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , Ratos , Ratos Nus , Ensaios Antitumorais Modelo de Xenoenxerto
5.
Sci Rep ; 11(1): 1191, 2021 01 13.
Artigo em Inglês | MEDLINE | ID: mdl-33441943

RESUMO

The in vivo assessment of tissue metabolism represents a novel strategy for the evaluation of oncologic disease. Hepatocellular carcinoma (HCC) is a high-prevalence, high-mortality tumor entity often discovered at a late stage. Recent evidence indicates that survival differences depend on metabolic alterations in tumor tissue, with particular focus on glucose metabolism and lactate production. Here, we present an in vivo imaging technique for metabolic tumor phenotyping in rat models of HCC. Endogenous HCC was induced in Wistar rats by oral diethyl-nitrosamine administration. Peak lactate-to-alanine signal ratios (L/A) were assessed with hyperpolarized magnetic resonance spectroscopic imaging (HPMRSI) after [1-13C]pyruvate injection. Cell lines were derived from a subset of primary tumors, re-implanted in nude rats, and assessed in vivo with dynamic hyperpolarized magnetic resonance spectroscopy (HPMRS) after [1-13C]pyruvate injection and kinetic modelling of pyruvate metabolism, taking into account systemic lactate production and recirculation. For ex vivo validation, enzyme activity and metabolite concentrations were spectroscopically quantified in cell and tumor tissue extracts. Mean peak L/A was higher in endogenous HCC compared to non-tumorous tissue. Dynamic HPMRS revealed higher pyruvate-to-lactate conversion rates (kpl) and lactate signal in subcutaneous tumors derived from high L/A tumor cells, consistent with ex vivo measurements of higher lactate dehydrogenase (LDH) levels in these cells. In conclusion, HPMRS and HPMRSI reveal distinct tumor phenotypes corresponding to differences in glycolytic metabolism in HCC tumor tissue.


Assuntos
Isótopos de Carbono/administração & dosagem , Carcinoma Hepatocelular/metabolismo , Neoplasias Hepáticas/metabolismo , Imageamento por Ressonância Magnética/métodos , Espectroscopia de Ressonância Magnética/métodos , Ácido Pirúvico/administração & dosagem , Alanina/metabolismo , Animais , Linhagem Celular Tumoral , Glicólise/fisiologia , L-Lactato Desidrogenase/metabolismo , Ácido Láctico/metabolismo , Masculino , Ratos , Ratos Nus , Ratos Wistar
6.
Nat Commun ; 12(1): 92, 2021 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-33397920

RESUMO

Telomere maintenance is a universal hallmark of cancer. Most tumors including low-grade oligodendrogliomas use telomerase reverse transcriptase (TERT) expression for telomere maintenance while astrocytomas use the alternative lengthening of telomeres (ALT) pathway. Although TERT and ALT are hallmarks of tumor proliferation and attractive therapeutic targets, translational methods of imaging TERT and ALT are lacking. Here we show that TERT and ALT are associated with unique 1H-magnetic resonance spectroscopy (MRS)-detectable metabolic signatures in genetically-engineered and patient-derived glioma models and patient biopsies. Importantly, we have leveraged this information to mechanistically validate hyperpolarized [1-13C]-alanine flux to pyruvate as an imaging biomarker of ALT status and hyperpolarized [1-13C]-alanine flux to lactate as an imaging biomarker of TERT status in low-grade gliomas. Collectively, we have identified metabolic biomarkers of TERT and ALT status that provide a way of integrating critical oncogenic information into non-invasive imaging modalities that can improve tumor diagnosis and treatment response monitoring.


Assuntos
Neoplasias Encefálicas/genética , Homeostase do Telômero , Telômero/metabolismo , Alanina/metabolismo , Animais , Biomarcadores Tumorais/metabolismo , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Isótopos de Carbono/metabolismo , Linhagem Celular Tumoral , Engenharia Genética , Glioma/genética , Glioma/metabolismo , Glioma/patologia , Ácido Láctico/metabolismo , Masculino , Metaboloma , Modelos Biológicos , Gradação de Tumores , Proteínas de Neoplasias/metabolismo , Espectroscopia de Prótons por Ressonância Magnética , Ácido Pirúvico/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos Nus , Telomerase/genética , Telomerase/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
7.
Am J Physiol Heart Circ Physiol ; 320(1): H458-H468, 2021 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-33095054

RESUMO

Coronary artery spasm (CAS) is an intense vasoconstriction of coronary arteries that causes total or subtotal vessel occlusion. The cardioprotective effect of sirtuin-1 (SIRT1) has been extensively highlighted in coronary artery diseases. The aims within this study include the investigation of the molecular mechanism by which SIRT1 alleviates CAS. SIRT1 expression was first determined by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and Western blot analysis in an endothelin-1 (ET-1)-induced rat CAS model. Interaction among SIRT1, nuclear factor-kappaB (NF-κB), myosin light chain kinase/myosin light chain-2 (MLCK/MLC2), and ET-1 was analyzed using luciferase reporter assay, RT-qPCR, and Western blot analysis. After ectopic expression and depletion experiments in vascular smooth muscle cells (VSMCs), contraction and proliferation of VSMCs and expression of contraction-related proteins (α-SMA, calponin, and SM22α) were measured by collagen gel contraction, 5-ethynyl-2'-deoxyuridine (EdU) assay, RT-qPCR, and Western blot analysis. The obtained results showed that SIRT1 expression was reduced in rat CAS models. However, overexpression of SIRT1 inhibited the contraction and proliferation of VSMCs in vitro. Mechanistic investigation indicated that SIRT1 inhibited NF-κB expression through deacetylation. Moreover, NF-κB could activate the MLCK/MLC2 pathway and upregulate ET-1 expression by binding to their promoter regions, thus inducing VSMC contraction and proliferation in vitro. In vivo experimental results also revealed that SIRT1 alleviated CAS through regulation of the NF-κB/MLCK/MLC2/ET-1 signaling axis. Collectively, our data suggested that SIRT1 could mediate the deacetylation of NF-κB, disrupt the MLCK/MLC2 pathway, and inhibit the expression of ET-1 to relieve CAS, providing a theoretical basis for the prospect of CAS treatment and prevention.NEW & NOTEWORTHY Rat coronary artery spasm models exhibit reduced expression of SIRT1. Overexpression of SIRT1 inhibits contraction and proliferation of VSMCs. SIRT1 inhibits NF-κB through deacetylation to modulate VSMC contraction and proliferation. NF-κB activates the MLCK/MLC2 pathway. NF-κB upregulates ET-1 to modulate VSMC contraction and proliferation.


Assuntos
Miosinas Cardíacas/metabolismo , Vasoespasmo Coronário/prevenção & controle , Endotelina-1/metabolismo , Músculo Liso Vascular/enzimologia , Cadeias Leves de Miosina/metabolismo , Quinase de Cadeia Leve de Miosina/metabolismo , NF-kappa B/metabolismo , Sirtuína 1/metabolismo , Vasoconstrição , Acetilação , Animais , Proliferação de Células , Forma Celular , Células Cultivadas , Vasoespasmo Coronário/enzimologia , Vasoespasmo Coronário/genética , Vasoespasmo Coronário/fisiopatologia , Vasos Coronários/enzimologia , Vasos Coronários/fisiopatologia , Modelos Animais de Doenças , Masculino , Músculo Liso Vascular/fisiopatologia , NF-kappa B/genética , Ratos Nus , Ratos Sprague-Dawley , Transdução de Sinais , Sirtuína 1/genética
8.
Chem Biol Interact ; 334: 109354, 2021 Jan 25.
Artigo em Inglês | MEDLINE | ID: mdl-33309620

RESUMO

Lactosyl-Sepharose binding proteins (LSBPs) were recently described in human pancreatic ductal adenocarcinoma (PDAC) Suit2-007 cells regarding their lectin-like properties and role in metastasis. This study further investigated how calcium and galactose influence the binding of LSBPs to the lactosyl resin as well as their anti-proliferative effect in Suit2-007 cells. Altered binding of LSBPs to the lactosyl resin was evaluated by affinity chromatography and mass spectrometry. Calcium binding EF-hand proteins were aligned and identified with a motif derived from the Uniprot protein database. The antiproliferative effects of LSBPs and monosaccharides were determined by MTT assay. In addition, LSBPs and galactose effects were investigated by chip array and tumor take in nude rats. LSBPs reduced Suit2-007 cells' proliferation with an IC50 of 125 µg/mL. Coincubation of LSBPs with EGTA decreased the number of LSBPs binding to the lactosyl resin by ~50%. Ca2+ -sensitive LSBPs included subgroups of galactose-sensitive (10%) and EF-hand calcium binding motifs containing (2.5%) proteins. In vitro, the combination of LSBPs with monosaccharides including galactose synergistically decreased cell proliferation compared to single agents (p < 0.05). In addition, LSBPs in combination with galactose prevented the tumor growth of Suit2-007 cells in nude rats, as opposed to single treatments. At mRNA level, the combination treatment modulated 5% of Ca2+ -sensitive LSBPs and downregulated 216 genes, 18% of which were up-regulated during PDAC progression. This study highlights the importance of calcium and galactose in modulating the affinity and anti-proliferative activity of LSBPs and their potential application as therapeutic agents for metastatic PDAC.


Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Cálcio/metabolismo , Proliferação de Células/fisiologia , Galactose/metabolismo , Ligação Proteica/fisiologia , Sefarose/metabolismo , Animais , Linhagem Celular Tumoral , Regulação para Baixo/fisiologia , Regulação Neoplásica da Expressão Gênica/fisiologia , Humanos , Lectinas/metabolismo , Masculino , RNA Mensageiro/metabolismo , Ratos , Ratos Nus , Regulação para Cima/fisiologia
9.
PLoS One ; 15(12): e0243156, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33264355

RESUMO

Young triple negative breast cancer (TNBC) patients are at high risk for developing very aggressive brain metastases associated with a poor prognosis and a high mortality rate. Preclinical models that allow follow-up by magnetic resonance imaging (MRI) can contribute to the development of new therapeutic approaches for brain metastasis. To date, preclinical brain tumor research has almost exclusively relied on xenograft mouse models. Yet, rats are an ideal model for imaging of brain metastasis as their larger brain offers better relative spatial resolution compared to a mouse brain. For the development of a clinically relevant rat model for TNBC brain metastasis, the MDA-MB-231br/eGFP cancer cell line can be used. However, as a result of species-dependent extracranial features, the propensity of the MDA-MB-231br/eGFP cancer cell line to metastasize exclusively to the brain needs to be enhanced by in vivo selection. In this study, repeated sequential passages of metastatic cancer cells obtained from brain metastases in nude rats were performed. Brain metastasis formation was evaluated using preclinical MRI, while bone metastasis formation was assessed using high-resolution computed tomography (CT) and 2-deoxy-2-[18F] fluoro-D-glucose ([18F] FDG) positron emission tomography (PET) imaging. Our results demonstrated that the metastatic tumor burden in the rat brain (number and volume) significantly increased with increasing passage, while the metastatic tumor burden in the skeleton (i.e., number of metastasis-affected bones) significantly decreased with increasing passage. However, bone metastasis development was not reduced to a negligible amount. Consequently, despite in vivo selection, our rat model is not recommended for investigating brain metastasis as a single disease. Our findings highlight the importance of well-reasoned selection of both the preclinical model and the cancer cell line in order to obtain reliable and reproducible scientific results.


Assuntos
Neoplasias Encefálicas/diagnóstico por imagem , Neoplasias Encefálicas/secundário , Proteínas de Fluorescência Verde/metabolismo , Inoculações Seriadas/métodos , Neoplasias de Mama Triplo Negativas/diagnóstico por imagem , Animais , Neoplasias Ósseas/diagnóstico por imagem , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/secundário , Neoplasias Encefálicas/metabolismo , Linhagem Celular Tumoral , Feminino , Fluordesoxiglucose F18/metabolismo , Proteínas de Fluorescência Verde/genética , Humanos , Imageamento por Ressonância Magnética , Transplante de Neoplasias , Ratos , Ratos Nus , Neoplasias de Mama Triplo Negativas/metabolismo
10.
Elife ; 92020 10 12.
Artigo em Inglês | MEDLINE | ID: mdl-33044169

RESUMO

Tissue resident mesenchymal stem/stromal cells (MSCs) occupy perivascular spaces. Profiling human adipose perivascular mesenchyme with antibody arrays identified 16 novel surface antigens, including endolysosomal protein CD107a. Surface CD107a expression segregates MSCs into functionally distinct subsets. In culture, CD107alow cells demonstrate high colony formation, osteoprogenitor cell frequency, and osteogenic potential. Conversely, CD107ahigh cells include almost exclusively adipocyte progenitor cells. Accordingly, human CD107alow cells drove dramatic bone formation after intramuscular transplantation in mice, and induced spine fusion in rats, whereas CD107ahigh cells did not. CD107a protein trafficking to the cell surface is associated with exocytosis during early adipogenic differentiation. RNA sequencing also suggested that CD107alow cells are precursors of CD107ahigh cells. These results document the molecular and functional diversity of perivascular regenerative cells, and show that relocation to cell surface of a lysosomal protein marks the transition from osteo- to adipogenic potential in native human MSCs, a population of substantial therapeutic interest.


Assuntos
Adipogenia/genética , Diferenciação Celular/genética , Proteína 1 de Membrana Associada ao Lisossomo/genética , Células-Tronco Mesenquimais/metabolismo , Osteogênese/genética , Adipócitos/metabolismo , Animais , Humanos , Proteína 1 de Membrana Associada ao Lisossomo/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Ratos , Ratos Nus , Células-Tronco/metabolismo
11.
J Bone Joint Surg Am ; 102(24): e135, 2020 Dec 16.
Artigo em Inglês | MEDLINE | ID: mdl-33079897

RESUMO

BACKGROUND: Despite the extensive use of cellular bone matrices (CBMs) in spine surgery, there is little evidence to support the contribution of cells within CBMs to bone formation. The objective of this study was to determine the contribution of cells to spinal fusion by direct comparisons among viable CBMs, devitalized CBMs, and cell-free demineralized bone matrix (DBM). METHODS: Three commercially available grafts were tested: a CBM containing particulate DBM (CBM-particulate), a CBM containing DBM fibers (CBM-fiber), and a cell-free product with DBM fibers only (DBM-fiber). CBMs were used in viable states (CBM-particulatev and CBM-fiberv) and devitalized (lyophilized) states (CBM-particulated and CBM-fiberd), resulting in 5 groups. Viable cell counts and bone morphogenetic protein-2 (BMP-2) content on enzyme-linked immunosorbent assay (ELISA) within each graft material were measured. A single-level posterolateral lumbar fusion was performed on 45 athymic rats with 3 lots of each product implanted into 9 animals per group. After 6 weeks, fusion was assessed using manual palpation, micro-computed tomography (µ-CT), and histological analysis. RESULTS: The 2 groups with viable cells were comparable with respect to cell counts, and pairwise comparisons showed no significant differences in BMP-2 content across the 5 groups. Manual palpation demonstrated fusion rates of 9 of 9 in the DBM-fiber specimens, 9 of 9 in the CBM-fiberd specimens, 8 of 9 in the CBM-fiberv specimens, and 0 of 9 in both CBM-particulate groups. The µ-CT maturity grade was significantly higher in the DBM-fiber group (2.78 ± 0.55) compared with the other groups (p < 0.0001), while none of the CBM-particulate samples demonstrated intertransverse fusion in qualitative assessments. The viable and devitalized samples in each CBM group were comparable with regard to fusion rates, bone volume fraction, µ-CT maturity grade, and histological features. CONCLUSIONS: The cellular component of 2 commercially available CBMs yielded no additional benefits in terms of spinal fusion. Meanwhile, the groups with a fiber-based DBM demonstrated significantly higher fusion outcomes compared with the CBM groups with particulate DBM, indicating that the DBM component is probably the key determinant of fusion. CLINICAL RELEVANCE: Data from the current study demonstrate that cells yielded no additional benefit in spinal fusion and emphasize the need for well-designed clinical studies on cellular graft materials.


Assuntos
Matriz Óssea/transplante , Fusão Vertebral/métodos , Animais , Matriz Óssea/química , Matriz Óssea/citologia , Proteína Morfogenética Óssea 2/análise , Contagem de Células , Sobrevivência Celular , Ensaio de Imunoadsorção Enzimática , Vértebras Lombares/diagnóstico por imagem , Vértebras Lombares/patologia , Vértebras Lombares/cirurgia , Masculino , Radiografia , Ratos , Ratos Nus , Microtomografia por Raio-X
12.
Cancer Res ; 80(22): 4986-4997, 2020 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-32973082

RESUMO

FGFR signaling is deregulated in many human cancers, and FGFR is considered a valid target in FGFR-deregulated tumors. Here, we examine the preclinical profile of futibatinib (TAS-120; 1-[(3S)-[4-amino-3-[(3,5-dimethoxyphenyl)ethynyl]-1H-pyrazolo[3, 4-d] pyrimidin-1-yl]-1-pyrrolidinyl]-2-propen-1-one), a structurally novel, irreversible FGFR1-4 inhibitor. Among a panel of 296 human kinases, futibatinib selectively inhibited FGFR1-4 with IC50 values of 1.4 to 3.7 nmol/L. Futibatinib covalently bound the FGFR kinase domain, inhibiting FGFR phosphorylation and, in turn, downstream signaling in FGFR-deregulated tumor cell lines. Futibatinib exhibited potent, selective growth inhibition of several tumor cell lines (gastric, lung, multiple myeloma, bladder, endometrial, and breast) harboring various FGFR genomic aberrations. Oral administration of futibatinib led to significant dose-dependent tumor reduction in various FGFR-driven human tumor xenograft models, and tumor reduction was associated with sustained FGFR inhibition, which was proportional to the administered dose. The frequency of appearance of drug-resistant clones was lower with futibatinib than a reversible ATP-competitive FGFR inhibitor, and futibatinib inhibited several drug-resistant FGFR2 mutants, including the FGFR2 V565I/L gatekeeper mutants, with greater potency than any reversible FGFR inhibitors tested (IC50, 1.3-50.6 nmol/L). These results indicate that futibatinib is a novel orally available, potent, selective, and irreversible inhibitor of FGFR1-4 with a broad spectrum of antitumor activity in cell lines and xenograft models. These findings provide a strong rationale for testing futibatinib in patients with tumors oncogenically driven by FGFR genomic aberrations, with phase I to III trials ongoing. SIGNIFICANCE: Preclinical characterization of futibatinib, an irreversible FGFR1-4 inhibitor, demonstrates selective and potent antitumor activity against FGFR-deregulated cancer cell lines and xenograft models, supporting clinical evaluation in patients with FGFR-driven tumors. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/22/4986/F1.large.jpg.


Assuntos
Antineoplásicos/uso terapêutico , Drogas em Investigação/uso terapêutico , Proteínas de Neoplasias/antagonistas & inibidores , Neoplasias/tratamento farmacológico , Pirazóis/uso terapêutico , Pirimidinas/uso terapêutico , Pirróis/uso terapêutico , Receptores de Fatores de Crescimento de Fibroblastos/antagonistas & inibidores , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/metabolismo , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Resistencia a Medicamentos Antineoplásicos , Drogas em Investigação/administração & dosagem , Drogas em Investigação/metabolismo , Neoplasias do Endométrio/tratamento farmacológico , Neoplasias do Endométrio/genética , Neoplasias do Endométrio/metabolismo , Feminino , Xenoenxertos , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Camundongos Nus , Camundongos SCID , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/genética , Mieloma Múltiplo/metabolismo , Proteínas de Neoplasias/metabolismo , Transplante de Neoplasias , Neoplasias/metabolismo , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/uso terapêutico , Pirazóis/farmacologia , Pirimidinas/farmacologia , Pirróis/farmacologia , Ratos , Ratos Nus , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/antagonistas & inibidores , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/genética , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/antagonistas & inibidores , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/genética , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/antagonistas & inibidores , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/genética , Receptor Tipo 4 de Fator de Crescimento de Fibroblastos/antagonistas & inibidores , Receptor Tipo 4 de Fator de Crescimento de Fibroblastos/genética , Receptores de Fatores de Crescimento de Fibroblastos/genética , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Neoplasias Gástricas/tratamento farmacológico , Neoplasias Gástricas/genética , Neoplasias Gástricas/metabolismo
13.
J Am Heart Assoc ; 9(16): e015841, 2020 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-32783519

RESUMO

Background Extracellular matrix, especially laminin-221, may play crucial roles in viability and survival of human-induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs) after in vivo transplant. Then, we hypothesized laminin-221 may have an adjuvant effect on therapeutic efficacy by enhancing cell viability and survival after transplantation of 3-dimensional engineered cardiac tissue (ECT) to a rat model of myocardial infarction. Methods and Results In vitro study indicates the impacts of laminin-221 on hiPS-CMs were analyzed on the basis of mechanical function, mitochondrial function, and tolerance to hypoxia. We constructed 3-dimensional ECT containing hiPS-CMs and fibrin gel conjugated with laminin-221. Heart function and in vivo behavior were assessed after engraftment of 3-dimensional ECT (laminin-conjugated ECT, n=10; ECT, n=10; control, n=10) in a rat model of myocardial infarction. In vitro assessment indicated that laminin-221 improves systolic velocity, diastolic velocity, and maximum capacity of oxidative metabolism of hiPS-CMs. Cell viability and lactate dehydrogenase production revealed that laminin-221 improved tolerance to hypoxia. Furthermore, analysis of mRNA expression revealed that antiapoptotic genes were upregulated in the laminin group under hypoxic conditions. Left ventricular ejection fraction of the laminin-conjugated ECT group was significantly better than that of other groups 4 weeks after transplantation. Laminin-conjugated ECT transplantation was associated with significant improvements in expression levels of rat vascular endothelial growth factor. In early assessments, cell survival was also improved in laminin-conjugated ECTs compared with ECT transplantation without laminin-221. Conclusions In vitro laminin-221 enhanced mechanical and metabolic function of hiPS-CMs and improved the therapeutic impact of 3-dimensional ECT in a rat ischemic cardiomyopathy model. These findings suggest that adjuvant laminin-221 may provide a clinical benefit to hiPS-CM constructs.


Assuntos
Sobrevivência Celular , Células-Tronco Pluripotentes Induzidas/citologia , Laminina/farmacologia , Infarto do Miocárdio/terapia , Miócitos Cardíacos/efeitos dos fármacos , Engenharia Tecidual , Animais , Apoptose/genética , Hipóxia Celular/efeitos dos fármacos , Modelos Animais de Doenças , Regulação da Expressão Gênica , Transplante de Coração , Humanos , Células-Tronco Pluripotentes Induzidas/fisiologia , L-Lactato Desidrogenase/biossíntese , Masculino , Contração Miocárdica/fisiologia , Miócitos Cardíacos/fisiologia , Miócitos Cardíacos/transplante , Neovascularização Fisiológica , RNA Mensageiro/metabolismo , Ratos , Ratos Nus , Proteínas Recombinantes/farmacologia , Volume Sistólico , Engenharia Tecidual/métodos , Regulação para Cima , Fator A de Crescimento do Endotélio Vascular/metabolismo , Remodelação Ventricular
14.
Sci Rep ; 10(1): 12794, 2020 07 30.
Artigo em Inglês | MEDLINE | ID: mdl-32732907

RESUMO

Articular cartilage damage does not heal spontaneously and causes joint dysfunction. The implantation of induced pluripotent stem cell (iPSC)-derived cartilage (iPS-Cart) is one candidate treatment to regenerate the damaged cartilage. However, concerns of tumorigenicity are associated with iPS-Cart, because the iPSC reprogramming process and long culture time for cartilage induction could increase the chance of malignancy. We evaluated the tumorigenic risks of iPS-Cart using HeLa cells as the reference. Spike tests revealed that contamination with 100 HeLa cells in 150 mg of iPS-Cart accelerated the cell growth rate. On the other hand, 150 mg of iPS-Cart without HeLa cells reached growth arrest and senescence after culture, suggesting less than 100 tumorigenic cells, assuming they behave like HeLa cells, contaminated iPS-Cart. The implantation of 10,000 or fewer HeLa cells into joint surface defects in the knee joint of nude rat did not cause tumor formation. These in vitro and in vivo studies collectively suggest that the implantation of 15 g or less iPS-Cart in the knee joint does not risk tumor formation if assuming that the tumorigenic cells in iPS-Cart are equivalent to HeLa cells and that nude rat knee joints are comparable to human knee joints in terms of tumorigenicity. However, considering the limited immunodeficiency of nude rats, the clinical amount of iPS-Cart for implantation needs to be determined cautiously.


Assuntos
Carcinogênese , Cartilagem Articular/patologia , Células-Tronco Pluripotentes Induzidas/patologia , Animais , Subpopulações de Linfócitos B , Cartilagem Articular/citologia , Proliferação de Células , Células HeLa , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Articulação do Joelho/citologia , Articulação do Joelho/patologia , Ratos Nus , Risco
15.
Viruses ; 12(7)2020 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-32630296

RESUMO

The Asian musk shrew (shrew) is a new reservoir of a rat hepatitis E virus (HEV) that has been classified into genotype HEV-C1 in the species Orthohepevirus C. However, there is no information regarding classification of the new rat HEV based on the entire genome sequences, and it remains unclear whether rat HEV transmits from shrews to humans. We herein inoculated nude rats (Long-Evans rnu/rnu) with a serum sample from a shrew trapped in China, which was positive for rat HEV RNA, to isolate and characterize the rat HEV distributed in shrews. A rat HEV strain, S1129, was recovered from feces of the infected nude rat, indicating that rat HEV was capable of replicating in rats. S1129 adapted and grew well in PLC/PRF/5 cells, and the recovered virus (S1129c1) infected Wistar rats. The entire genomes of S1129 and S1129c1 contain four open reading frames and share 78.3-81.8% of the nucleotide sequence identities with known rat HEV isolates, demonstrating that rat HEVs are genetically diverse. We proposed that genotype HEV-C1 be further classified into subtypes HEV-C1a to HEV-C1d and that the S1129 strain circulating in the shrew belonged to the new subtype HEV-C1d. Further studies should focus on whether the S1129 strain infects humans.


Assuntos
Vírus da Hepatite E/classificação , Hepatite E/virologia , Musaranhos/virologia , Animais , China , Fezes/virologia , Feminino , Genoma Viral , Genótipo , Vírus da Hepatite E/isolamento & purificação , Filogenia , Ratos/virologia , Ratos Long-Evans , Ratos Nus , Ratos Wistar , Replicação Viral
16.
Nat Commun ; 11(1): 3369, 2020 07 06.
Artigo em Inglês | MEDLINE | ID: mdl-32632153

RESUMO

Induced pluripotent stem cell (iPSC)-derived dopaminergic (DA) neurons are an expected source for cell-based therapies for Parkinson's disease (PD). The regulatory criteria for the clinical application of these therapies, however, have not been established. Here we show the results of our pre-clinical study, in which we evaluate the safety and efficacy of dopaminergic progenitors (DAPs) derived from a clinical-grade human iPSC line. We confirm the characteristics of DAPs by in vitro analyses. We also verify that the DAP population include no residual undifferentiated iPSCs or early neural stem cells and have no genetic aberration in cancer-related genes. Furthermore, in vivo studies using immunodeficient mice reveal no tumorigenicity or toxicity of the cells. When the DAPs are transplanted into the striatum of 6-OHDA-lesioned rats, the animals show behavioral improvement. Based on these results, we started a clinical trial to treat PD patients in 2018.


Assuntos
Neurônios Dopaminérgicos/citologia , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Neurais/transplante , Doença de Parkinson/terapia , Transplante de Células-Tronco/métodos , Animais , Diferenciação Celular/genética , Linhagem Celular , Modelos Animais de Doenças , Dopamina/metabolismo , Neurônios Dopaminérgicos/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Macaca fascicularis , Masculino , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Avaliação de Resultados em Cuidados de Saúde/métodos , Avaliação de Resultados em Cuidados de Saúde/estatística & dados numéricos , Ratos Nus , Transplante Heterólogo
17.
Commun Biol ; 3(1): 330, 2020 06 25.
Artigo em Inglês | MEDLINE | ID: mdl-32587337

RESUMO

Volumetric muscle loss (VML) is the traumatic or surgical loss of skeletal muscle beyond the inherent regenerative capacity of the body, generally leading to severe functional deficit. Formation of appropriate somato-motor innervations remains one of the biggest challenges for both autologous grafts as well as tissue-engineered muscle constructs. We aim to address this challenge by developing pre-innervated tissue-engineered muscle comprised of long aligned networks of spinal motor neurons and skeletal myocytes on aligned nanofibrous scaffolds. Motor neurons led to enhanced differentiation and maturation of skeletal myocytes in vitro. These pre-innervated tissue-engineered muscle constructs when implanted in a rat VML model significantly increased satellite cell density, neuromuscular junction maintenance, graft revascularization, and muscle volume over three weeks as compared to myocyte-only constructs and nanofiber scaffolds alone. These pro-regenerative effects may enhance functional neuromuscular regeneration following VML, thereby improving the levels of functional recovery following these devastating injuries.


Assuntos
Músculo Esquelético/fisiologia , Atrofia Muscular/terapia , Engenharia Tecidual/métodos , Animais , Contagem de Células , Sobrevivência Celular , Microambiente Celular , Masculino , Camundongos , Neurônios Motores/fisiologia , Células Musculares/fisiologia , Músculo Esquelético/inervação , Junção Neuromuscular/fisiologia , Ratos , Ratos Nus , Regeneração , Tecidos Suporte
18.
Acta Biochim Pol ; 67(2): 149-155, 2020 Jun 08.
Artigo em Inglês | MEDLINE | ID: mdl-32506868

RESUMO

The present investigation aimed at developing Doxorubicin (DOX)-loaded liposome-mediated drug delivery system for head and neck cancer. The liposomes were prepared by film hydration technique using egg phosphatidylcholine and cholesterol using Box-Behnken statistical design. The prepared liposomes were evaluated for the percentage encapsulation efficiency, particle size and in vitro release. The average particle size of the DOX-encapsulating liposomes formulated by thin-film hydration technique was between 150.5 nm and 200 nm with an average particle size of 165.80 nm. The PDI (Polydispersity index) was found to be 0.315 which indicated that particles were monodispersed and narrow-dispersed. In vitro drug release of DOX-loaded liposomes and DOX-loaded peptide-conjugated liposomes was performed in phosphate buffered saline (pH 7.4) and both formulations showed sustained release behavior over the period of 40 hours. The optimized liposomal formulation was conjugated to a peptide and subsequently radiolabeled with 186Re-perrhenate solution and BMEDA-glucoheptonate-stannous chloride solution. Comparative cytotoxicity assay of DOX, DOX-liposomes and DOX-liposomes-peptide on SCC9 cells was performed and it was found that liposomal formulation was not cytotoxic. The antitumor efficacy of 186Re-liposomes, unlabelled liposomes, 186Re-perrhenate solution and 186Re-BMEDA solution was determined in SCC cell lines injected into BALB/c-nu/nu athymic nude rats. The efficacy of antitumor activity was found to be in the following order: peptide-conjugated DOX-loaded liposomes>unlabelled liposomes>186Re-perrhenate solution>186Re-BMEDA solution. The present investigation showed that peptide-conjugated DOX-loaded liposomes significantly suppress the tumor growth in the nude rat model. These results suggest the significant potential of liposomes as carriers for clinical applications in head and neck cancer.


Assuntos
Antibióticos Antineoplásicos/administração & dosagem , Doxorrubicina/administração & dosagem , Sistemas de Liberação de Medicamentos/métodos , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto/métodos , Animais , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Liberação Controlada de Fármacos , Neoplasias de Cabeça e Pescoço/patologia , Humanos , Lipossomos , Masculino , Tamanho da Partícula , Peptídeos/química , Ratos , Ratos Nus , Resultado do Tratamento
19.
Biopharm Drug Dispos ; 41(7): 283-294, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32562504

RESUMO

TAK-448 is the investigational metastin/kisspeptin analog, which is known to have an anti-tumor effect through suppression of androgen hormones (luteinizing hormone and testosterone) levels. This study developed pharmacokinetic-pharmacodynamic (PK/PD) models of TAK-448 and leuprorelin acetate (TAP-144) in a rat vertebral-cancer of the prostate (VCaP) androgen-sensitive prostate cancer xenograft model to quantitatively assess and compare the anti-tumor effects of both drugs. A potential contribution of the hormone-independent direct effects of TAK-448 to the tumor growth inhibition was also investigated in the in vivo rat xenograft model, because our in vitro experiments revealed that TAK-448 may also directly suppress VCaP cellular proliferation. The PK/PD model successfully described the time course of tumor growth inhibition after drug treatment as well as the development of resistance to the inhibition of androgen hormones, following drug treatment or castration. The EC50 of the hormone-dependent inhibitory effect of TAK-448 was much lower than that of TAP-144, and TAK-448 also has a faster onset of anti-tumor effect than TAP-144, demonstrating that TAK-448 has a stronger overall anti-tumor effect than TAP-144. In addition, model inference, by incorporating a hormone-independent inhibition pathway of TAK-448 into the PK-PD model, suggested that such a direct inhibition pathway for TAK-448 cannot be excluded, as also indicated by in vitro studies, but its EC50 would be approximately three orders of magnitude higher than that of the hormone-dependent pathway. This study helps to understand the potential and mechanism of TAK-448 as a prostate cancer treatment.


Assuntos
Antineoplásicos Hormonais/farmacologia , Kisspeptinas/farmacologia , Modelos Biológicos , Neoplasias da Próstata/tratamento farmacológico , Animais , Antineoplásicos Hormonais/farmacocinética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Humanos , Kisspeptinas/farmacocinética , Leuprolida/farmacologia , Masculino , Neoplasias da Próstata/patologia , Ratos , Ratos Nus , Fatores de Tempo , Ensaios Antitumorais Modelo de Xenoenxerto
20.
PLoS One ; 15(6): e0234087, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32511282

RESUMO

BACKGROUND: Ventricular septal perforation and left ventricular aneurysm are examples of potentially fatal complications of myocardial infarction. While various artificial materials are used in the repair of these issues, the possibility of associated infection and calcification is non-negligible. Cell-seeded biodegradable tissue-engineered patches may be a potential solution. This study evaluated the feasibility of a new left ventricular patch rat model to study neotissue formation in biodegradable cardiac patches. METHODS: Human induced pluripotent stem cell-derived cardiac progenitor cells (hiPS-CPCs) were cultured onto biodegradable patches composed of polyglycolic acid and a 50:50 poly (l-lactide-co-ε-caprolactone) copolymer for one week. After culturing, patches were implanted into left ventricular walls of male athymic rats. Unseeded controls were also used (n = 10/group). Heart conditions were followed by echocardiography and patches were subsequently explanted at 1, 2, 6, and 9 months post-implantation for histological evaluation. RESULT: Throughout the study, no patches ruptured demonstrating the ability to withstand the high pressure left ventricular system. One month after transplantation, the seeded patch did not stain positive for human nuclei. However, many new blood vessels formed within patches with significantly greater vessels in the seeded group at the 6 month time point. Echocardiography showed no significant difference in left ventricular contraction rate between the two groups. Calcification was found inside patches after 6 months, but there was no significant difference between groups. CONCLUSION: We have developed a surgical method to implant a bioabsorbable scaffold into the left ventricular environment of rats with a high survival rate. Seeded hiPS-CPCs did not differentiate into cardiomyocytes, but the greater number of new blood vessels in seeded patches suggests the presence of cell seeding early in the remodeling process might provide a prolonged effect on neotissue formation. This experiment will contribute to the development of a treatment model for left ventricular failure using iPS cells in the future.


Assuntos
Células-Tronco Pluripotentes Induzidas/citologia , Miócitos Cardíacos/citologia , Engenharia Tecidual , Implantes Absorvíveis , Animais , Modelos Animais de Doenças , Ecocardiografia , Ventrículos do Coração/metabolismo , Ventrículos do Coração/patologia , Humanos , Masculino , Infarto do Miocárdio/patologia , Infarto do Miocárdio/terapia , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/transplante , Poliésteres/química , Ácido Poliglicólico/química , Ratos , Ratos Nus , Tecidos Suporte/química , Troponina T/metabolismo , Função Ventricular
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