RESUMEN
BACKGROUND: Pathogenic variants in SCN5A can result in long QT syndrome type 3, a life-threatening genetic disease. Adenine base editors can convert targeted A T base pairs to G C base pairs, offering a promising tool to correct pathogenic variants. METHODS: We generated a long QT syndrome type 3 mouse model by introducing the T1307M pathogenic variant into the Scn5a gene. The adenine base editor was split into 2 smaller parts and delivered into the heart by adeno-associated virus serotype 9 (AAV9-ABEmax) to correct the T1307M pathogenic variant. RESULTS: Both homozygous and heterozygous T1307M mice showed significant QT prolongation. Carbachol administration induced Torsades de Pointes or ventricular tachycardia for homozygous T1307M mice (20%) but not for heterozygous or wild-type mice. A single intraperitoneal injection of AAV9-ABEmax at postnatal day 14 resulted in up to 99.20% Scn5a transcripts corrected in T1307M mice. Scn5a mRNA correction rate >60% eliminated QT prolongation; Scn5a mRNA correction rate <60% alleviated QT prolongation. Partial Scn5a correction resulted in cardiomyocytes heterogeneity, which did not induce severe arrhythmias. We did not detect off-target DNA or RNA editing events in ABEmax-treated mouse hearts. CONCLUSIONS: These findings show that in vivo AAV9-ABEmax editing can correct the variant Scn5a allele, effectively ameliorating arrhythmia phenotypes. Our results offer a proof of concept for the treatment of hereditary arrhythmias.
Asunto(s)
Trastorno del Sistema de Conducción Cardíaco , Edición Génica , Síndrome de QT Prolongado , Ratones , Animales , Síndrome de QT Prolongado/genética , Síndrome de QT Prolongado/terapia , Síndrome de QT Prolongado/diagnóstico , Arritmias Cardíacas , Miocitos Cardíacos , Adenina , ARN Mensajero , Canal de Sodio Activado por Voltaje NAV1.5/genética , MutaciónRESUMEN
Hydrogels are attractive materials for tissue engineering, but efforts to date have shown limited ability to produce the microstructural features necessary to promote cellular self-organization into hierarchical three-dimensional (3D) organ models. Here we develop a hydrogel ink containing prefabricated gelatin fibres to print 3D organ-level scaffolds that recapitulate the intra- and intercellular organization of the heart. The addition of prefabricated gelatin fibres to hydrogels enables the tailoring of the ink rheology, allowing for a controlled sol-gel transition to achieve precise printing of free-standing 3D structures without additional supporting materials. Shear-induced alignment of fibres during ink extrusion provides microscale geometric cues that promote the self-organization of cultured human cardiomyocytes into anisotropic muscular tissues in vitro. The resulting 3D-printed ventricle in vitro model exhibited biomimetic anisotropic electrophysiological and contractile properties.
Asunto(s)
Gelatina , Andamios del Tejido , Humanos , Andamios del Tejido/química , Gelatina/química , Miocitos Cardíacos , Ingeniería de Tejidos/métodos , Hidrogeles/química , Impresión TridimensionalRESUMEN
BACKGROUND: RBPs (RNA-binding proteins) perform indispensable functions in the post-transcriptional regulation of gene expression. Numerous RBPs have been implicated in cardiac development or physiology based on gene knockout studies and the identification of pathogenic RBP gene mutations in monogenic heart disorders. The discovery and characterization of additional RBPs performing indispensable functions in the heart will advance basic and translational cardiovascular research. METHODS: We performed a differential expression screen in zebrafish embryos to identify genes enriched in nkx2.5-positive cardiomyocytes or cardiopharyngeal progenitors compared to nkx2.5-negative cells from the same embryos. We investigated the myocardial-enriched gene RNA-binding protein with multiple splicing (variants) 2 [RBPMS2)] by generating and characterizing rbpms2 knockout zebrafish and human cardiomyocytes derived from RBPMS2-deficient induced pluripotent stem cells. RESULTS: We identified 1848 genes enriched in the nkx2.5-positive population. Among the most highly enriched genes, most with well-established functions in the heart, we discovered the ohnologs rbpms2a and rbpms2b, which encode an evolutionarily conserved RBP. Rbpms2 localizes selectively to cardiomyocytes during zebrafish heart development and strong cardiomyocyte expression persists into adulthood. Rbpms2-deficient embryos suffer from early cardiac dysfunction characterized by reduced ejection fraction. The functional deficit is accompanied by myofibril disarray, altered calcium handling, and differential alternative splicing events in mutant cardiomyocytes. These phenotypes are also observed in RBPMS2-deficient human cardiomyocytes, indicative of conserved molecular and cellular function. RNA-sequencing and comparative analysis of genes mis-spliced in RBPMS2-deficient zebrafish and human cardiomyocytes uncovered a conserved network of 29 ortholog pairs that require RBPMS2 for alternative splicing regulation, including RBFOX2, SLC8A1, and MYBPC3. CONCLUSIONS: Our study identifies RBPMS2 as a conserved regulator of alternative splicing, myofibrillar organization, and calcium handling in zebrafish and human cardiomyocytes.
Asunto(s)
Calcio , Miocardio , Proteínas de Unión al ARN , Proteínas de Pez Cebra , Animales , Humanos , Calcio/metabolismo , Miocardio/metabolismo , Miocitos Cardíacos/metabolismo , Proteínas Represoras/metabolismo , Factores de Empalme de ARN/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Pez Cebra/genética , Pez Cebra/metabolismo , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismoRESUMEN
Z-discs are core ultrastructural organizers of cardiomyocytes that modulate many facets of cardiac pathogenesis. Yet a comprehensive proteomic atlas of Z-disc-associated components remain incomplete. Here, we established an adeno-associated virus (AAV)-delivered, cardiomyocyte-specific, proximity-labeling approach to characterize the Z-disc proteome in vivo. We found palmdelphin (PALMD) as a novel Z-disc-associated protein in both adult murine cardiomyocytes and human pluripotent stem cell-derived cardiomyocytes. Germline and cardiomyocyte-specific Palmd knockout mice were grossly normal at baseline but exhibited compromised cardiac hypertrophy and aggravated cardiac injury upon long-term isoproterenol treatment. By contrast, cardiomyocyte-specific PALMD overexpression was sufficient to mitigate isoproterenol-induced cardiac injury. PALMD ablation perturbed the transverse tubule (T-tubule)-sarcoplasmic reticulum (SR) ultrastructures, which formed the Z-disc-associated junctional membrane complex (JMC) essential for calcium handling and cardiac function. These phenotypes were associated with the reduction of nexilin (NEXN), a crucial Z-disc-associated protein that is essential for both Z-disc and JMC structures and functions. PALMD interacted with NEXN and enhanced its protein stability while the Nexn mRNA level was not affected. AAV-based NEXN addback rescued the exacerbated cardiac injury in isoproterenol-treated PALMD-depleted mice. Together, this study discovered PALMD as a potential target for myocardial protection and highlighted in vivo proximity proteomics as a powerful approach to nominate novel players regulating cardiac pathogenesis.
RESUMEN
Bone is one of the prone metastatic sites of patients with advanced breast cancer. The "vicious cycle" between osteoclasts and breast cancer cells plays an essential role in osteolytic bone metastasis from breast cancer. In order to inhibit bone metastasis from breast cancer, NIR-II photoresponsive bone-targeting nanosystems (CuP@PPy-ZOL NPs) are designed and synthesized. CuP@PPy-ZOL NPs can trigger the photothermal-enhanced Fenton response and photodynamic effect to enhance the photothermal treatment (PTT) effect and thus achieve synergistic anti-tumor effect. Meanwhile, they exhibit a photothermal enhanced ability to inhibit osteoclast differentiation and promote osteoblast differentiation, which reshaped the bone microenvironment. CuP@PPy-ZOL NPs effectively inhibited the proliferation of tumor cells and bone resorption in the in vitro 3D bone metastases model of breast cancer. In a mouse model of breast cancer bone metastasis, CuP@PPy-ZOL NPs combined with PTT with NIR-II significantly inhibited the tumor growth of breast cancer bone metastases and osteolysis while promoting bone repair to achieve the reversal of osteolytic breast cancer bone metastases. Furthermore, the potential biological mechanisms of synergistic treatment are identified by conditioned culture experiments and mRNA transcriptome analysis. The design of this nanosystem provides a promising strategy for treating osteolytic bone metastases.
Asunto(s)
Neoplasias Óseas , Osteólisis , Animales , Ratones , Terapia Fototérmica , Microambiente Tumoral , Huesos/patología , Neoplasias Óseas/terapia , Neoplasias Óseas/patología , Osteoclastos , Osteólisis/terapia , Osteólisis/patología , Línea Celular TumoralRESUMEN
The detrimental impact of organic contaminants on optical components poses a significant obstacle to high-energy laser systems. However, irregularities or defects on the surface of optical components during manufacturing can affect the process of organic contaminant removal. Thus, a comprehensive understanding of the intricate interplay among surface roughness, contaminant absorption, and ablation is essential to effectively address the challenges of laser-induced damage. In this study, a molecular dynamics approach was employed to investigate the interaction between laser-fused silica and contaminants and to analyze the influence of surface roughness on the removal of contaminants from fused silica. Research findings demonstrate that during laser irradiation, organic contaminants on the surface of mechanical components diffuse into the optical elements. As the laser flux increases, the contaminants gradually decompose into smaller molecular clusters. Additionally, the phenomenon of contaminant ablation is observed to consist of two distinct phases: the "Thermal expansion phase" and the "Thermal ablation phase." The study examines the impact of substrate roughness on the contaminant removal in these two phases. It is found that a higher surface roughness leads to stronger thermal expansion and vaporization of contaminants. With increasing roughness of the fused silica substrate, the corresponding van der Waals energy and pressure decrease under the same laser fluence, making the removal of contaminants easier. These results provide valuable insights into the interaction between laser irradiation and organic contaminants.
RESUMEN
[Figure: see text].
Asunto(s)
Cardiomiopatía Hipertrófica/terapia , Terapia Genética/métodos , Cadenas Pesadas de Miosina/genética , Animales , Cardiomiopatía Hipertrófica/genética , Células Cultivadas , Edición Génica/métodos , Células HEK293 , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Mutación , Cadenas Pesadas de Miosina/metabolismoRESUMEN
Photosensitizers play a key role in bioimaging and photodynamic therapy (PDT) of cancer. However, conventional photosensitizers usually do not achieve the desired efficacy in PDT due to their poor photostability, targeting ability, and responsiveness. Herein, we designed a series of photosensitizers with aggregation-induced emission (AIE) effect using benzothiazole- triphenylamine (BZT-triphenylamine) as the parent nucleus. The synthesized compound SIN ((E)-2-(4-(diphenylamino)styryl)-3-(4-iodobutyl)benzo[d]thiazol-3-ium) exhibits good biocompatibility, photostability, and bright emission in the near-infrared range (600-800 nm). The fluorescence emission intensity is responsive to viscosity, with significant fluorescence enhancement (48 times) and high fluorescence quantum yield (4.45 %) at high viscosity. Moreover, SIN has particular lysosome targeting properties with a Pearson correlation coefficient (PCC) of 0.97 and has good 1O2 generation ability under white light irradiation, especially in a weak acidic environment. Thus, SIN can realize good bioimaging ability and photodynamic therapeutic efficacy under the highly viscous and weakly acidic environment of lysosomes in the tumor cells. This study indicates that SIN has potential as a multifunctional organic photosensitizer for bioimaging and PDT of tumor.
Asunto(s)
Neoplasias , Fotoquimioterapia , Humanos , Fármacos Fotosensibilizantes/farmacología , Fármacos Fotosensibilizantes/uso terapéutico , Fotoquimioterapia/métodos , Neoplasias/diagnóstico por imagen , Neoplasias/tratamiento farmacológico , Luz , LisosomasRESUMEN
BACKGROUND: Mutations in tafazzin (TAZ), a gene required for biogenesis of cardiolipin, the signature phospholipid of the inner mitochondrial membrane, causes Barth syndrome (BTHS). Cardiomyopathy and risk of sudden cardiac death are prominent features of BTHS, but the mechanisms by which impaired cardiolipin biogenesis causes cardiac muscle weakness and arrhythmia are poorly understood. METHODS: We performed in vivo electrophysiology to define arrhythmia vulnerability in cardiac-specific TAZ knockout mice. Using cardiomyocytes derived from human induced pluripotent stem cells and cardiac-specific TAZ knockout mice as model systems, we investigated the effect of TAZ inactivation on Ca2+ handling. Through genome editing and pharmacology, we defined a molecular link between TAZ mutation and abnormal Ca2+ handling and contractility. RESULTS: A subset of mice with cardiac-specific TAZ inactivation developed arrhythmias, including bidirectional ventricular tachycardia, atrial tachycardia, and complete atrioventricular block. Compared with wild-type controls, BTHS-induced pluripotent stem cell-derived cardiomyocytes had increased diastolic Ca2+ and decreased Ca2+ transient amplitude. BTHS-induced pluripotent stem cell-derived cardiomyocytes had higher levels of mitochondrial and cellular reactive oxygen species than wild-type controls, which activated CaMKII (Ca2+/calmodulin-dependent protein kinase II). Activated CaMKII phosphorylated the RYR2 (ryanodine receptor 2) on serine 2814, increasing Ca2+ leak through RYR2. Inhibition of this reactive oxygen species-CaMKII-RYR2 pathway through pharmacological inhibitors or genome editing normalized aberrant Ca2+ handling in BTHS-induced pluripotent stem cell-derived cardiomyocytes and improved their contractile function. Murine Taz knockout cardiomyocytes also exhibited elevated diastolic Ca2+ and decreased Ca2+ transient amplitude. These abnormalities were ameliorated by Ca2+/calmodulin-dependent protein kinase II or reactive oxygen species inhibition. CONCLUSIONS: This study identified a molecular pathway that links TAZ mutation with abnormal Ca2+ handling and decreased cardiomyocyte contractility. This pathway may offer therapeutic opportunities to treat BTHS and potentially other diseases with elevated mitochondrial reactive oxygen species production.
Asunto(s)
Síndrome de Barth/genética , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Calcio/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Animales , Síndrome de Barth/fisiopatología , Humanos , Ratones , Ratones NoqueadosRESUMEN
Rhodopsin mutation and misfolding is a common cause of autosomal dominant retinitis pigmentosa (RP). Using a luciferase reporter assay, we undertook a small-molecule high-throughput screening (HTS) of 68, 979 compounds and identified nine compounds that selectively reduced the misfolded P23H rhodopsin without an effect on the wild type (WT) rhodopsin protein. Further, we found five of these compounds, including methotrexate (MTX), promoted P23H rhodopsin degradation that also cleared out other misfolded rhodopsin mutant proteins. We showed MTX increased P23H rhodopsin degradation via the lysosomal but not the proteasomal pathway. Importantly, one intravitreal injection (IVI) of 25 pmol MTX increased electroretinogram (ERG) response and rhodopsin level in the retinae of RhoP23H/+ knock-in mice at 1 month of age. Additionally, four weekly IVIs increased the photoreceptor cell number in the retinae of RhoP23H/+ mice compared to vehicle control. Our study indicates a therapeutic potential of repurposing MTX for the treatment of rhodopsin-associated RP.
Asunto(s)
Retinitis Pigmentosa/metabolismo , Rodopsina/metabolismo , Animales , Línea Celular , Electrorretinografía/métodos , Femenino , Células HEK293 , Humanos , Masculino , Ratones , Proteínas Mutantes/metabolismo , Mutación/genética , Células 3T3 NIH , Células Fotorreceptoras/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Pliegue de Proteína , Retina/metabolismo , Retinitis Pigmentosa/genética , Rodopsina/genéticaRESUMEN
Organic small molecules with near-infrared (NIR) absorption hold great promise as the phototheranostic agents for clinical translation by virtue of their inherent merits such as well-defined chemical structure, high purity and good reproducibility. Probes that happen to be based on cyanine dyes exhibit strong NIR-absorbing and efficient photothermal conversion, representing a new class of photothermal agents (PAs) for photothermal therapy (PTT), and taking into account the heat susceptibility of Mitochondria (Mito), we designed and prepared a mitochondria-targeted organic small molecule (Mito-BWQ) based on thiazole orange maternal unit that can effectively kill tumor cells through the hyperpyrexia generated in the lesions under exogenous laser irradiation. The Confocal laser scanning microscope was employed to determine the preferential targeting of Mito-BWQ to the mitochondria of MCF-7 cells and U87 cells. When subjected to 600 nm laser radiation, Mito-BWQ produced an increase in temperature in test systems and this increase was dependent on both the laser power and probe concentration. In vitro tests, cytotoxicity was observed when cells were incubated with Mito-BWQ and exposed to laser irradiation. The PTT in vivo also showed that Mito-BWQ performed remarkably in tumor inhibition. This study thus provides a vital starting point for the creation of thiazole orange-based PTT formulations and promotes further advances in the field of PAs-based anticancer research and therapy.
Asunto(s)
Antineoplásicos/farmacología , Benzotiazoles/farmacología , Mitocondrias/efectos de los fármacos , Terapia Fototérmica , Quinolinas/farmacología , Animales , Antineoplásicos/síntesis química , Antineoplásicos/química , Benzotiazoles/síntesis química , Benzotiazoles/química , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Humanos , Neoplasias Mamarias Experimentales/tratamiento farmacológico , Neoplasias Mamarias Experimentales/metabolismo , Neoplasias Mamarias Experimentales/patología , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Mitocondrias/metabolismo , Estructura Molecular , Quinolinas/síntesis química , Quinolinas/química , Relación Estructura-Actividad , Células Tumorales CultivadasRESUMEN
BACKGROUND: Modeling of human arrhythmias with induced pluripotent stem cell-derived cardiomyocytes has focused on single-cell phenotypes. However, arrhythmias are the emergent properties of cells assembled into tissues, and the impact of inherited arrhythmia mutations on tissue-level properties of human heart tissue has not been reported. METHODS: Here, we report an optogenetically based, human engineered tissue model of catecholaminergic polymorphic ventricular tachycardia (CPVT), an inherited arrhythmia caused by mutation of the cardiac ryanodine channel and triggered by exercise. We developed a human induced pluripotent stem cell-derived cardiomyocyte-based platform to study the tissue-level properties of engineered human myocardium. We investigated pathogenic mechanisms in CPVT by combining this novel platform with genome editing. RESULTS: In our model, CPVT tissues were vulnerable to developing reentrant rhythms when stimulated by rapid pacing and catecholamine, recapitulating hallmark features of the disease. These conditions elevated diastolic Ca2+ levels and increased temporal and spatial dispersion of Ca2+ wave speed, creating a vulnerable arrhythmia substrate. Using Cas9 genome editing, we pinpointed a single catecholamine-driven phosphorylation event, ryanodine receptor-serine 2814 phosphorylation by Ca2+/calmodulin-dependent protein kinase II, that is required to unmask the arrhythmic potential of CPVT tissues. CONCLUSIONS: Our study illuminates the molecular and cellular pathogenesis of CPVT and reveals a critical role of calmodulin-dependent protein kinase II-dependent reentry in the tissue-scale mechanism of this disease. We anticipate that this approach will be useful for modeling other inherited and acquired cardiac arrhythmias.
Asunto(s)
Células Madre Pluripotentes Inducidas/fisiología , Miocitos Cardíacos/patología , Miocitos Cardíacos/fisiología , Taquicardia Ventricular/patología , Taquicardia Ventricular/fisiopatología , Ingeniería de Tejidos/métodos , Potenciales de Acción/fisiología , Células Cultivadas , Humanos , Células Madre Pluripotentes Inducidas/química , Miocitos Cardíacos/química , Optogenética/métodosRESUMEN
Long noncoding RNAs (lncRNAs), by virtue of their versatility and multilevel gene regulation, have emerged as attractive pharmacological targets for treating heterogeneous and complex malignancies like triple-negative breast cancer (TNBC). Despite multiple studies on lncRNA functions in tumor pathology, systemic targeting of these "undruggable" macromolecules with conventional approaches remains a challenge. Here, we demonstrate effective TNBC therapy by nanoparticle-mediated RNAi of the oncogenic lncRNA DANCR, which is significantly overexpressed in TNBC. Tumor-targeting RGD-PEG-ECO/siDANCR nanoparticles were formulated via self-assembly of multifunctional amino lipid ECO, cyclic RGD peptide-PEG, and siDANCR for systemic delivery. MDA-MB-231 and BT549 cells treated with the therapeutic RGD-PEG-ECO/siDANCR nanoparticles exhibited 80-90% knockdown in the expression of DANCR for up to 7 days, indicating efficient intracellular siRNA delivery and sustained target silencing. The RGD-PEG-ECO/siDANCR nanoparticles mediated excellent in vitro therapeutic efficacy, reflected by significant reduction in the invasion, migration, survival, tumor spheroid formation, and proliferation of the TNBC cell lines. At the molecular level, functional ablation of DANCR dynamically impacted the oncogenic nexus by downregulating PRC2-mediated H3K27-trimethylation and Wnt/EMT signaling, and altering the phosphorylation profiles of several kinases in the TNBC cells. Furthermore, systemic administration of the RGD-PEG-ECO/siDANCR nanoparticles at a dose of 1 mg/kg siRNA in nude mice bearing TNBC xenografts resulted in robust suppression of TNBC progression with no overt toxic side-effects, underscoring the efficacy and safety of the nanoparticle therapy. These results demonstrate that nanoparticle-mediated modulation of onco-lncRNAs and their molecular targets is a promising approach for developing curative therapies for TNBC and other cancers.
Asunto(s)
Terapia Genética , Nanopartículas , ARN Largo no Codificante/antagonistas & inhibidores , ARN Interferente Pequeño/administración & dosificación , Neoplasias de la Mama Triple Negativas/terapia , Animales , Línea Celular Tumoral , Femenino , Humanos , Ratones , Ratones Desnudos , ARN Interferente Pequeño/química , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Importin 13 (IPO13) is a key member of the importin ß superfamily, which can transport cargoes both into and out of the nucleus to contribute to a variety of important cellular processes. IPO13 is known to undergo phosphorylation, but the impact of this on function has not been investigated. Here, we show for the first time that IPO13 is phosphorylated by cAMP-dependent protein kinase A specifically at serine 193. Results from fluorescence recovery after photobleaching and fluorescence loss in photobleaching approaches establish that negative charge at serine 193 through phosphorylation or point mutation both reduces IPO13 nuclear import and increases its nuclear export. Importantly, phosphorylation also appears to enhance cargo interaction on the part of IPO13, with significant impact on localization, as shown for the Pax6 homeobox-containing transcription partner. This is the first report that IPO13 can be phosphorylated at Ser193 and that this modification regulates IPO13 subcellular localization and nucleocytoplasmic transport function, with important implications for IPO13's role in development and other processes.
Asunto(s)
Núcleo Celular/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Citoplasma/metabolismo , Carioferinas/metabolismo , Transporte Activo de Núcleo Celular/fisiología , Núcleo Celular/genética , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Citoplasma/genética , Células HeLa , Humanos , Carioferinas/genética , Factor de Transcripción PAX6/genética , Factor de Transcripción PAX6/metabolismo , Fosforilación/fisiologíaRESUMEN
Integrins, a large family of cell adhesion receptors, have been shown to play an important role for glioma proliferation and invasion. Several integrin receptors, including αvß3, αvß5, and α5ß1, have generated clinical interest for glioma diagnosis and antitumor therapy. Integrin α5ß1 has been highlighted as a prognostic and diagnostic marker in glioma, and its expression is correlated with a worse prognosis in high-grade glioma. However, unlike extensively studied integrins αvß3 and αvß5, very few integrin α5ß1-specific radiotracers have been reported. Developing α5ß1-specific radiotracers may provide alternative diagnosis and evaluation options in addition to well-studied αvß3/αvß5-specific tracers, and they may add new documents for profiling tumor progression. Here, a novel integrin α5ß1-specific probe (99m)Tc-HisoDGR was fabricated for SPECT (single-photon emission computed tomography) imaging of glioma. To confirm its selective targeting of integrin α5ß1 in vivo, the mouse models of α5ß1-positive U87MG human glioma were subjected to SPECT/CT scans, and biodistribution experiments and blocking studies were performed. Small-animal SPECT/CT imaging experiments demonstrated that the tumors were clearly visualized in both subcutaneous and orthotopic glioma tumor models with clear background at 0.5, 1, and 2 h p.i. The tumor accumulation of (99m)Tc-HisoDGR showed significant reduction when excess cold isoDGR peptide was coinjected, suggesting that the tumor uptake was specifically mediated. Our work revealed that (99m)Tc-HisoDGR represented a powerful molecular probe for integrin α5ß1-positive cancer imaging; moreover, it might be a promising tool for evaluating malignancy, predicting prognosis, selecting subpopulations of patients who might be sensitive to integrin α5ß1-targeted drugs, and assessing and monitoring the response to integrin α5ß1-targeted drugs in clinical trials.
Asunto(s)
Glioma/diagnóstico por imagen , Glioma/patología , Integrina alfa5beta1/metabolismo , Oligopéptidos/química , Oligopéptidos/metabolismo , Compuestos de Organotecnecio/química , Tomografía Computarizada de Emisión de Fotón Único/métodos , Animales , Línea Celular Tumoral , Transformación Celular Neoplásica , Regulación Neoplásica de la Expresión Génica , Glioma/metabolismo , Humanos , Ratones , Radioquímica , Especificidad por Sustrato , Distribución TisularRESUMEN
During the last decade, the usage of silver nanoparticles in biomedical fields has increased rapidly, mainly due to their excellent antibacterial effects. They are used in many medical products such as wound dressings, catheters, bone cement and artificial cardiac valves. In tissue engineering, silver nanoparticles are often loaded as a filler for fabrication of nanocomposite scaffolds which subsequently are seeded with human mesenchymal stem cells. Thus, possible adverse effects of silver nanoparticles on human stem cells should be investigated carefully to ensure a safe usage. In this study, silver nanoparticles with a mean diameter of ~30 nm were prepared and their toxicity in human mesenchymal stem cells was investigated. Transmission electron microscopic images reveal the uptake and localization of the silver nanoparticles in the cytoplasm. Upon internalization of Ag NPs inside the cells, an increase in the release of lactate dehydrogenase and the production of reactive oxygen species was quantified. Furthermore, they caused a reduction in both cell viability and mitochondrial membrane potential in a dose-dependent manner. Annexin V-FITC/PI staining implied that silver nanoparticles did not only induce apoptosis but also cause necrosis. Based on cell cycle analysis, G2/M arrest was detected in cells treated with silver nanoparticles, implicating DNA damage. The high level of reactive oxygen species induced by nanoparticles is considered to be the main cause of their toxicity.
Asunto(s)
Células de la Médula Ósea/metabolismo , Citotoxinas , Puntos de Control de la Fase G2 del Ciclo Celular/efectos de los fármacos , Puntos de Control de la Fase M del Ciclo Celular/efectos de los fármacos , Células Madre Mesenquimatosas/metabolismo , Nanopartículas del Metal , Plata , Células de la Médula Ósea/citología , Citotoxinas/efectos adversos , Citotoxinas/química , Citotoxinas/farmacología , Humanos , Células Madre Mesenquimatosas/citología , Nanopartículas del Metal/efectos adversos , Nanopartículas del Metal/química , Nanopartículas del Metal/ultraestructura , Plata/efectos adversos , Plata/química , Plata/farmacologíaRESUMEN
To study the mechanisms underlying the IL-6-promoted angiogenic microenvironment in EGFRvIII-positive glioblastoma, VEGF expression in EGFRvIII-positive/negative tumors was determined by optical molecular imaging. Next, the HUVEC tube formation assay, Western blot, qPCR, RNA silencing, chromatin immunoprecipitation, luciferase reporter and ELISA assays were performed to examine the role of IL-6 and C/EBPß in the formation of the angiogenic microenvironment in EGFRvIII-positive tumors. Finally, in vitro and in vivo genistein treatment experiments were conducted to challenge the interaction between the IL-6 promoter and C/EBPß. Optical imaging revealed greater VEGF expression in EGFRvIII-positive tumor-bearing mice, suggesting an angiogenic microenvironment. In vitro experiments demonstrated that C/EBPß-mediated regulation of IL-6 was indispensable for maintenance of this angiogenic microenvironment. In contrast, genistein-mediated upregulation of CHOP impeded C/EBPß interaction with the IL-6 promoter, thus disturbing the angiogenic microenvironment. This more malignant microenvironment in EGFRvIII glioblastoma is generated, at least in part, by greater VEGF, IL-6 and C/EBPß expression. Interaction of C/EBPß with the IL-6 promoter maintains this angiogenic microenvironment, while disturbance of this dynamically balanced interaction inhibits EGFRvIII tumor proliferation by reducing both VEGF and IL-6 expression.
Asunto(s)
Proteína beta Potenciadora de Unión a CCAAT/metabolismo , Receptores ErbB/metabolismo , Genisteína/farmacología , Glioblastoma/metabolismo , Interleucina-6/metabolismo , Factor de Transcripción CHOP/metabolismo , Animales , Proteína beta Potenciadora de Unión a CCAAT/genética , Línea Celular Tumoral , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana , Humanos , Interleucina-6/antagonistas & inhibidores , Ratones , Ratones Endogámicos BALB C , Trasplante de Neoplasias , Neovascularización Patológica , Regiones Promotoras Genéticas , Microambiente TumoralRESUMEN
ß-Emitters can produce Cerenkov radiation that is detectable by Cerenkov luminescence imaging (CLI), allowing the combination of PET and CLI with one radiotracer for both tumor diagnosis and visual guidance during surgery. Recently, the clinical feasibility of CLI with the established therapeutic reagent Na(131)I and the PET tracer (18)F-FDG was demonstrated. (68)Ga possesses a higher Cerenkov light output than (18)F and (131)I, which would result in higher sensitivity for CLI and improve the outcome of CLI in clinical applications. However, the research on (68)Ga-based tumor-specific tracers for CLI is limited. In this study, we examined the use of (68)Ga-radiolabeled DOTA-3PRGD2 ((68)Ga-3PRGD2) for dual PET and CLI of orthotopic U87MG human glioblastoma. For this purpose, the Cerenkov efficiencies of (68)Ga and (18)F were measured with the IVIS Spectrum system (PerkinElmer, USA). The CLI signal intensity of (68)Ga was 15 times stronger than that of (18)F. PET and CLI of (68)Ga-3PRGD2 were performed in U87MG human glioblastoma xenografts. Both PET and CLI revealed a remarkable accumulation of (68)Ga-3PRGD2 in the U87MG human glioblastoma xenografts at 1 h p.i. with an extremely low background in the brain when compared with (18)F-FDG. Furthermore, (68)Ga-3PRGD2 was used for dual PET and CLI of orthotopic human glioblastoma. The orthotopic human glioblastoma was clearly visualized by both imaging modalities. In addition, the biodistribution of (68)Ga-3PRGD2 was assessed in normal mice to estimate the radiation dosimetry. The whole-body effective dose is 20.1 ± 3.3 µSv/MBq, which is equal to 3.7 mSv per whole-body PET scan with a 5 mCi injection dose. Thus, (68)Ga-3PRGD2 involves less radiation exposure in patients when compared with (18)F-FDG (7.0 mSv). The use of (68)Ga-3PRGD2 in dual PET and CLI shows great promise for tumor diagnosis and image-guided surgery.
Asunto(s)
Radioisótopos de Galio/química , Glioblastoma/diagnóstico , Oligopéptidos/química , Imagen Óptica/métodos , Compuestos Organometálicos/química , Péptidos Cíclicos/química , Tomografía de Emisión de Positrones/métodos , Animales , Línea Celular Tumoral , Femenino , Fluorodesoxiglucosa F18/farmacocinética , Radioisótopos de Galio/farmacocinética , Humanos , Luminiscencia , Mediciones Luminiscentes/métodos , Ratones , Ratones Endogámicos BALB C , Oligopéptidos/farmacocinética , Compuestos Organometálicos/farmacocinética , Péptidos Cíclicos/farmacocinética , Distribución TisularRESUMEN
Antiangiogenic therapy is an effective strategy to inhibit tumor growth. Endostar, as an approved antiangiogenesis agent, inhibits the newborn vascular endothelial cells, causing the decrease of integrin αvß3 expression. Radiolabeled 3PRGD2, a novel PEGlayted RGD dimer probe (PEG4-E[PEG4-c(RGDfK)]2) showed highly specific targeting capability to integrin αvß3, which could be used for monitoring the efficacy of Endostar antiangiogenic therapy. In this study, (68)Ga-3PRGD2 PET imaging was performed in Endostar treated/untreated Lewis Lung Carcinoma (LLC) mice on days 3, 7, 14, and 21 post-treatment for monitoring the tumor response to Endostar treatment, with the (18)F-FDG imaging as control. As a result, (68)Ga-3PRGD2 PET reflected the tumor response to Endostar antiangiogenic therapy much earlier (day 3 post-treatment vs day 14 post-treatment) and more accurately than that of (18)F-FDG metabolic imaging, which provides new opportunities to develop individualized therapeutic approaches, establish optimized dosages and dose intervals for effective treatment that improve the survival rate of patients.
Asunto(s)
Inhibidores de la Angiogénesis/química , Endostatinas/química , Radioisótopos de Galio , Neovascularización Patológica/tratamiento farmacológico , Oligopéptidos/química , Tomografía de Emisión de Positrones , Animales , Carcinoma Pulmonar de Lewis , Línea Celular Tumoral , Femenino , Radioisótopos de Galio/química , Humanos , Ratones , Ratones Endogámicos C57BL , Radiofármacos , Proteínas Recombinantes/química , Factores de TiempoRESUMEN
The DNAJC19 gene, a member of DNAJ heat shock protein (Hsp40) family, is localized within the inner mitochondrial membrane (IMM) and plays a crucial role in regulating the function and localization of mitochondrial Hsp70 (MtHsp70). Mutations in the DNAJC19 gene cause Dilated Cardiomyopathy with Ataxia Syndrome (DCMA). The precise mechanisms underlying the DCMA phenotype caused by DNAJC19 mutations remain poorly understood, and effective treatment modalities were lacking unitl recently. By using CRISPR-Cas9 gene editing technology, this study generated a DNAJC19-knockout (DNAJC19-KO) human embryonic stem cell line (hESC), which will be a useful tool in studying the pathogenesis of DCMA.