RESUMEN
Neurobiological consequences of traumatic brain injury (TBI) result from a complex interplay of secondary injury responses and sequela that mediates chronic disability. Endothelial cells are important regulators of the cerebrovascular response to TBI. Our work demonstrates that genetic deletion of endothelial cell (EC)-specific EPH receptor A4 (EphA4) using conditional EphA4f/f/Tie2-Cre and EphA4f/f/VE-Cadherin-CreERT2 knockout (KO) mice promotes blood-brain barrier (BBB) integrity and tissue protection, which correlates with improved motor function and cerebral blood flow recovery following controlled cortical impact (CCI) injury. scRNAseq of capillary-derived KO ECs showed increased differential gene expression of BBB-related junctional and actin cytoskeletal regulators, namely, A-kinase anchor protein 12, Akap12, whose presence at Tie2 clustering domains is enhanced in KO microvessels. Transcript and protein analysis of CCI-injured whole cortical tissue or cortical-derived ECs suggests that EphA4 limits the expression of Cldn5, Akt, and Akap12 and promotes Ang2. Blocking Tie2 using sTie2-Fc attenuated protection and reversed Akap12 mRNA and protein levels cortical-derived ECs. Direct stimulation of Tie2 using Vasculotide, angiopoietin-1 memetic peptide, phenocopied the neuroprotection. Finally, we report a noteworthy rise in soluble Ang2 in the sera of individuals with acute TBI, highlighting its promising role as a vascular biomarker for early detection of BBB disruption. These findings describe a contribution of the axon guidance molecule, EphA4, in mediating TBI microvascular dysfunction through negative regulation of Tie2/Akap12 signaling.
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Barrera Hematoencefálica , Lesiones Traumáticas del Encéfalo , Receptor EphA4 , Animales , Ratones , Proteínas de Anclaje a la Quinasa A/genética , Proteínas de Anclaje a la Quinasa A/metabolismo , Barrera Hematoencefálica/metabolismo , Lesiones Traumáticas del Encéfalo/genética , Lesiones Traumáticas del Encéfalo/metabolismo , Proteínas de Ciclo Celular/metabolismo , Células Endoteliales/metabolismo , Ratones Noqueados , Receptor TIE-2/genética , Receptor TIE-2/metabolismo , Receptor EphA4/genética , Receptor EphA4/metabolismoRESUMEN
In an effort to synthesize chemically recyclable thermoplastic elastomers, a redox-switchable catalytic system was developed to synthesize triblock copolymers containing stiff poly(lactic acid) (PLA) end blocks and a flexible poly(tetrahydrofuran-co-cyclohexene oxide) (poly(THF-co-CHO) copolymer as the mid-block. The orthogonal reactivity induced by changing the oxidation state of the iron-based catalyst enabled the synthesis of the triblock copolymers in a single reaction flask from a mixture of monomers. The triblock copolymers demonstrated improved flexibility compared to poly(l-lactic acid) (PLLA) and thermomechanical properties that resemble thermoplastic elastomers, including a rubbery plateau in the range of -60 to 40 °C. The triblock copolymers containing a higher percentage of THF versus CHO were more flexible, and a blend of triblock copolymers containing PLLA and poly(d-lactic acid) (PDLA) end-blocks resulted in a stereocomplex that further increased polymer flexibility. Besides the low cost of lactide and THF, the sustainability of this new class of triblock copolymers was also supported by their depolymerization, which was achieved by exposing the copolymers sequentially to FeCl3 and ZnCl2 /PEG under reactive distillation conditions.
RESUMEN
Hydrogen sulfide (H2S) is produced endogenously by several enzymatic pathways and modulates physiological functions in mammals. Quantification of H2S in biochemical systems remains challenging because of the presence of interferents with similar reactivity, particularly thiols. Herein, we present a new quantification method based on the formation of pyrene excimers in solution. We synthesized the probe 2-(maleimido)ethyl 4-pyrenylbutanoate (MEPB) and determined that MEPB reacted with H2S in a two-step reaction to yield the thioether-linked dimer (MEPB)2S, which formed excimers upon excitation, with a broad peak of fluorescence emission centered at 480 nm. In contrast, we found that the products formed with thiols showed peaks at 378 and 398 nm. The difference in emission between the products prevented the interference. Furthermore, we showed that the excimer fluorescence signal yielded a linear response to H2S, with a limit of detection of 54 nM in a fluorometer. Our quantification method with MEPB was successfully applied to follow the reaction of H2S with glutathione disulfide and to quantify the production of H2S from cysteine by Escherichia coli. In conclusion, this method represents an addition to the toolkit of biochemists to quantify H2S specifically and sensitively in biochemical systems.
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Colorantes Fluorescentes , Sulfuro de Hidrógeno , Pirenos , Cisteína , Colorantes Fluorescentes/química , Sulfuro de Hidrógeno/análisis , Sulfuro de Hidrógeno/química , Pirenos/química , Compuestos de Sulfhidrilo/química , FluorescenciaRESUMEN
BACKGROUND: Hydrogen sulfide (H2S) donors are crucial tools not only for understanding the role of H2S in cellular function but also as promising therapeutic agents for oxidative stress-related diseases. This study aimed to explore the effect of amino acid-derived N-thiocarboxyanhydrides (NTAs), which release physiological H2S levels in the presence of carbonic anhydrase, on porcine sperm function during short-term incubation with and without induced oxidative stress. For this purpose, we employed two H2S-releasing NTAs with release half-lives (t1/2) in the range of hours that derived from the amino acids glycine (Gly-NTA) or leucine (Leu-NTA). Because carbonic anhydrase is crucial for H2S release from NTAs, we first measured the activity of this enzyme in the porcine ejaculate. Then, we tested the effect of Gly- and Leu-NTAs at 10 and 1 nM on sperm mitochondrial activity, plasma membrane integrity, acrosomal status, motility, motile subpopulations, and redox balance during short-term incubation at 38 °C with and without a reactive oxygen species (ROS)-generating system. RESULTS: Our results show that carbonic anhydrase is found both in spermatozoa and seminal plasma, with activity notably higher in the latter. Both Gly- and Leu-NTAs did not exert any noxious effects, but they enhanced sperm mitochondrial activity in the presence and absence of oxidative stress. Moreover, NTAs (except for Leu-NTA 10 nM) tended to preserve the sperm redox balance against the injuries provoked by oxidative stress, which provide further support to the antioxidant effect of H2S on sperm function. Both compounds also increased progressive motility over short-term incubation, which may translate into prolonged sperm survival. CONCLUSIONS: The presence of carbonic anhydrase activity in mammalian spermatozoa makes NTAs promising molecules to investigate the role of H2S in sperm biology. For the first time, beneficial effects of NTAs on mitochondrial activity have been found in mammalian cells in the presence and absence of oxidative stress. NTAs are interesting compounds to investigate the role of H2S in sperm mitochondria-dependent events and to develop H2S-related therapeutic protocols against oxidative stress in assisted reproductive technologies.
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Aminoácidos , Anhidrasas Carbónicas , Masculino , Animales , Porcinos , Aminoácidos/metabolismo , Semillas/metabolismo , Espermatozoides , Estrés Oxidativo , Mitocondrias , Especies Reactivas de Oxígeno/metabolismo , Anhidrasas Carbónicas/metabolismo , Anhidrasas Carbónicas/farmacología , MamíferosRESUMEN
Antisense oligonucleotides (ASOs) have emerged as a new class of drugs to treat a wide range of diseases, including neurological indications. Spinraza, an ASO that modulates splicing of SMN2 RNA, has shown profound disease modifying effects in Spinal Muscular Atrophy (SMA) patients, energizing efforts to develop ASOs for other neurological diseases. While SMA specifically affects spinal motor neurons, other neurological diseases affect different central nervous system (CNS) regions, neuronal and non-neuronal cells. Therefore, it is important to characterize ASO distribution and activity in all major CNS structures and cell types to have a better understanding of which neurological diseases are amenable to ASO therapy. Here we present for the first time the atlas of ASO distribution and activity in the CNS of mice, rats, and non-human primates (NHP), species commonly used in preclinical therapeutic development. Following central administration of an ASO to rodents, we observe widespread distribution and target RNA reduction throughout the CNS in neurons, oligodendrocytes, astrocytes and microglia. This is also the case in NHP, despite a larger CNS volume and more complex neuroarchitecture. Our results demonstrate that ASO drugs are well suited for treating a wide range of neurological diseases for which no effective treatments are available.
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Sistema Nervioso Central/química , Ratones/metabolismo , Oligonucleótidos Antisentido/farmacocinética , Primates/metabolismo , Ratas/metabolismo , Animales , Sistema Nervioso Central/citología , Femenino , Hibridación in Situ , Inyecciones Intraventriculares , Inyecciones Espinales , Macaca fascicularis , Masculino , Neuroglía/química , Neuronas/química , Oligonucleótidos Antisentido/administración & dosificación , Especificidad de Órganos , ARN Largo no Codificante/análisis , ARN Largo no Codificante/antagonistas & inhibidores , ARN Largo no Codificante/genética , Ratas Sprague-Dawley , Ribonucleasa H , Distribución TisularRESUMEN
We report three constitutionally isomeric tetrapeptides, each comprising one glutamic acid (E) residue, one histidine (H) residue, and two lysine (KS ) residues functionalized with side-chain hydrophobic S-aroylthiooxime (SATO) groups. Depending on the order of amino acids, these amphiphilic peptides self-assembled in aqueous solution into different nanostructures:nanoribbons, a mixture of nanotoroids and nanoribbons, or nanocoils. Each nanostructure catalyzed hydrolysis of a model substrate, with the nanocoils exhibiting the greatest rate enhancement and the highest enzymatic efficiency. Coarse-grained molecular dynamics simulations, analyzed with unsupervised machine learning, revealed clusters of H residues in hydrophobic pockets along the outer edge of the nanocoils, providing insight for the observed catalytic rate enhancement. Finally, all three supramolecular nanostructures catalyzed hydrolysis of the l-substrate only when a pair of enantiomeric Boc-l/d-Phe-ONp substrates were tested. This study highlights how subtle molecular-level changes can influence supramolecular nanostructures, and ultimately affect catalytic efficiency.
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Nanoestructuras , Nanotubos de Carbono , Péptidos/química , Nanoestructuras/química , Isomerismo , CatálisisRESUMEN
Inducing high levels of reactive oxygen species (ROS) inside tumor cells is a cancer therapy method termed chemodynamic therapy (CDT). Relying on delivery of Fenton reaction promoters such as Fe2+ , CDT takes advantage of overproduced ROS in the tumor microenvironment. We developed a peptide-H2 S donor conjugate, complexed with Fe2+ , termed AAN-PTC-Fe2+ . The AAN tripeptide was specifically cleaved by legumain, an enzyme overexpressed in glioma cells, to release carbonyl sulfide (COS). Hydrolysis of COS by carbonic anhydrase formed H2 S, an inhibitor of catalase, an enzyme that detoxifies H2 O2 . Fe2+ and H2 S together increased intracellular ROS levels and decreased viability in C6 glioma cells compared with controls lacking either Fe2+ , the AAN sequence, or the ability to generate H2 S. AAN-PTC-Fe2+ performed better than temezolimide while exhibiting no cytotoxicity toward H9C2 cardiomyocytes. This study provides an H2 S-amplified, enzyme-responsive platform for synergistic cancer treatment.
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Glioma , Nanopartículas , Neoplasias , Humanos , Especies Reactivas de Oxígeno , Péptidos/farmacología , Línea Celular Tumoral , Microambiente Tumoral , Peróxido de HidrógenoRESUMEN
The optimal medical management of patients following endovascular deep venous interventions remains ill-defined. As such, the Society of Interventional Radiology Foundation (SIRF) convened a multidisciplinary group of experts in a virtual Research Consensus Panel (RCP) to develop a prioritized research agenda regarding antithrombotic therapy following deep venous interventions. The panelists presented the gaps in knowledge followed by discussion and ranking of research priorities based on clinical relevance, overall impact, and technical feasibility. The following research topics were identified as high priority: 1) characterization of biological processes leading to in-stent stenosis/rethrombosis; 2) identification and validation of methods to assess venous flow dynamics and their effect on stent failure; 3) elucidation of the role of inflammation and anti-inflammatory therapies; and 4) clinical studies to compare antithrombotic strategies and improve venous outcome assessment. Collaborative, multicenter research is necessary to answer these questions and thereby enhance the care of patients with venous disease.
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Radiología Intervencionista , Enfermedades Vasculares , Consenso , Humanos , Investigación , Enfermedades Vasculares/diagnóstico por imagen , Enfermedades Vasculares/terapia , Procedimientos Quirúrgicos VascularesRESUMEN
Endocytosis is a mechanism by which cells sense their environment and internalize various nutrients, growth factors and signaling molecules. This process initiates at the plasma membrane, converges with autophagy, and terminates at the lysosome. It is well-established that cellular uptake of antisense oligonucleotides (ASOs) proceeds through the endocytic pathway; however, only a small fraction escapes endosomal trafficking while the majority are rendered inactive in the lysosome. Since these pathways converge and share common molecular machinery, it is unclear if autophagy-related trafficking participates in ASO uptake or whether modulation of autophagy affects ASO activity and localization. To address these questions, we investigated the effects of autophagy modulation on ASO activity in cells and mice. We found that enhancing autophagy through small-molecule mTOR inhibition, serum-starvation/fasting, and ketogenic diet, increased ASO-mediated target reduction in vitro and in vivo. Additionally, autophagy activation enhanced the localization of ASOs into autophagosomes without altering intracellular concentrations or trafficking to other compartments. These results support a novel role for autophagy and the autophagosome as a previously unidentified compartment that participates in and contributes to enhanced ASO activity. Further, we demonstrate non-chemical methods to enhance autophagy and subsequent ASO activity using translatable approaches such as fasting or ketogenic diet.
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Autofagia/fisiología , Oligonucleótidos Antisentido/metabolismo , Animales , Autofagosomas/metabolismo , Transporte Biológico/fisiología , Células Cultivadas , Endocitosis/fisiología , Células HEK293 , Células HeLa , Humanos , Ratones , Ratones Endogámicos C57BL , Oligonucleótidos Antisentido/genética , Interferencia de ARN , Transducción de SeñalRESUMEN
Centronuclear myopathies (CNM) are a group of severe muscle diseases for which no effective therapy is currently available. We have previously shown that reduction of the large GTPase DNM2 in a mouse model of the X-linked form, due to loss of myotubularin phosphatase MTM1, prevents the development of the skeletal muscle pathophysiology. As DNM2 is mutated in autosomal dominant forms, here we tested whether DNM2 reduction can rescue DNM2-related CNM in a knock-in mouse harboring the p.R465W mutation (Dnm2RW/+) and displaying a mild CNM phenotype similar to patients with the same mutation. A single intramuscular injection of adeno-associated virus-shRNA targeting Dnm2 resulted in reduction in protein levels 5 wk post injection, with a corresponding improvement in muscle mass and fiber size distribution, as well as an improvement in histopathological CNM features. To establish a systemic treatment, weekly i.p. injections of antisense oligonucleotides targeting Dnm2 were administered to Dnm2RW/+mice for 5 wk. While muscle mass, histopathology, and muscle ultrastructure were perturbed in Dnm2RW/+mice compared with wild-type mice, these features were indistinguishable from wild-type mice after reducing DNM2. Therefore, DNM2 knockdown via two different strategies can efficiently correct the myopathy due to DNM2 mutations, and it provides a common therapeutic strategy for several forms of centronuclear myopathy. Furthermore, we provide an example of treating a dominant disease by targeting both alleles, suggesting that this strategy may be applied to other dominant diseases.
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Dinamina II/genética , Miopatías Estructurales Congénitas/genética , Animales , Masculino , Ratones , Ratones Endogámicos C57BL , Músculo Esquelético/metabolismo , Mutación/genética , Proteínas Tirosina Fosfatasas no Receptoras/genéticaRESUMEN
Persulfides (R-SSH) have been hypothesized as potent redox modulators and signaling compounds. Reported herein is the synthesis, characterization, and in vivo evaluation of a persulfide donor that releases N-acetyl cysteine persulfide (NAC-SSH) in response to the prokaryote-specific enzyme nitroreductase. The donor, termed NDP-NAC, decomposed in response to E.â coli nitroreductase, resulting in release of NAC-SSH. NDP-NAC elicited gastroprotective effects in mice that were not observed in animals treated with control compounds incapable of persulfide release or in animals treated with Na2 S. NDP-NAC induced these effects by the upregulation of beneficial small- and medium-chain fatty acids and through increasing growth of Turicibacter sanguinis, a beneficial gut bacterium. It also decreased the populations of Synergistales bacteria, opportunistic pathogens implicated in gastrointestinal infections. This study reveals the possibility of maintaining gut health or treating microbiome-related diseases by the targeted delivery of reactive sulfur species.
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Antibacterianos/farmacología , Microbioma Gastrointestinal/efectos de los fármacos , Profármacos/farmacología , Sulfuros/farmacología , Animales , Antibacterianos/síntesis química , Antibacterianos/química , Diseño de Fármacos , Escherichia coli/efectos de los fármacos , Cinética , Listeria monocytogenes/efectos de los fármacos , Ratones , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Profármacos/síntesis química , Profármacos/química , Staphylococcus aureus/efectos de los fármacos , Sulfuros/síntesis química , Sulfuros/químicaRESUMEN
Self-assembly of amphiphilic peptide-based building blocks gives rise to a plethora of interesting nanostructures such as ribbons, fibers, and tubes. However, it remains a great challenge to employ peptide self-assembly to directly produce nanostructures with lower symmetry than these highly symmetric motifs. We report here our discovery that persistent and regular crescent nanostructures with a diameter of 28 ± 3 nm formed from a series of tetrapeptides with the general structure AdKSKSEX (Ad = adamantyl group, KS = lysine residue functionalized with an S-aroylthiooxime (SATO) group, E = glutamic acid residue, and X = variable amino acid residue). In the presence of cysteine, the biological signaling gas hydrogen sulfide (H2S) was released from the SATO units of the crescent nanostructures, termed peptide-H2S donor conjugates (PHDCs), reducing levels of reactive oxygen species (ROS) in macrophage cells. Additional in vitro studies showed that the crescent nanostructures alleviated cytotoxicity induced by phorbol 12-myristate-13-acetate more effectively than common H2S donors and a PHDC of a similar chemical structure, AdKSKSE, that formed short nanoworms instead of nanocrescents. Cell internalization studies indicated that nanocrescent-forming PHDCs were more effective in reducing ROS levels in macrophages because they entered into and remained in cells better than nanoworms, highlighting how nanostructure morphology can affect bioactivity in drug delivery.
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Nanoestructuras/química , Oligopéptidos/química , Animales , Supervivencia Celular/efectos de los fármacos , Sulfuro de Hidrógeno/química , Sulfuro de Hidrógeno/metabolismo , Macrófagos/citología , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Ratones , Oligopéptidos/farmacología , Células RAW 264.7 , Especies Reactivas de Oxígeno/metabolismo , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
The plasmonic properties of silver nanoparticle (AgNP) arrays are directly controlled by AgNP size, shape, and spatial arrangement. Reported here is a strategy to prepare chiral AgNP arrays templated by two constitutionally isomeric aromatic peptide amphiphiles (APAs), KSC'EKS and C'EKSKS (KS = S-aroylthiooxime-modified lysine, C' = citrulline, and E = glutamic acid). In phosphate buffer, both APAs initially self-assembled into nanoribbons with a similar geometry. However, in the presence of silver ions and poly(sodium 4-styrenesulfonate) (PSSS), one of the nanoribbons (KSC'EKS) turned into nanohelices with a regular twisting pitch, while the other (C'EKSKS) remained as nanoribbons. Both were used as templates for synthesis of arrays of â¼8 nm AgNPs to understand how small changes in molecular structure affect the plasmonic properties of these chiral AgNP/APA hybrids. Both hybrids showed improved colloidal stability compared to pure AgNPs, and both showed enhanced sensitivity as surface-enhanced Raman spectroscopy (SERS) substrates for model analytes, with nanohelices showing better SERS performance compared to their nanoribbon counterparts and pure AgNPs.
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Nanopartículas del Metal/química , Péptidos/síntesis química , Plata/química , Estructura Molecular , Tamaño de la Partícula , Péptidos/química , Propiedades de SuperficieRESUMEN
Controlled release is an essential requirement for delivery of hydrogen sulfide (H2S) because of its reactive nature, short half-life in biological fluids, and toxicity at high concentrations. In this context, H2S delivery via hydrogels may be beneficial as they can deliver H2S locally at the site of interest. Herein, we employed hydrogels based on aromatic peptide amphiphiles (APAs) with tunable mechanical properties to modulate the rates of H2S release. The APAs contained an aromatic S-aroylthiooxime (SATO) H2S donor attached with a linker to a short IAVEEE hexapeptide. Linker units included carbonyl, substituted O-methylenes, alkenyl, and alkyl segments with the goal of evaluating the role of linker structure on self-assembly, capacity for hydrogelation, and H2S release rate. We studied each peptide by transmission electron microscopy, circular dichroism spectroscopy, and rheology, and we measured H2S release rates from each gel, triggering SATO decomposition and release of H2S by addition of cysteine (Cys). Using an H2S-selective electrode probe as well as a turn-on fluorescent H2S probe in the presence of H9C2 cardiomyocytes, we found that the rate of H2S release from the hydrogels depended on the rate of Cys penetration into the nanofiber core with stiffer gels showing longer overall release.
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Sulfuro de Hidrógeno , Nanofibras , Semivida , Hidrogeles , PéptidosRESUMEN
Despite the widespread use of hydrogels in biomedical applications, little is known regarding the effect of crosslinker topology on hydrogel degradation. Dendritic and linear elastin-like peptides (ELPs) were used as crosslinkers for hyaluronic acid (HA) hydrogels, and their enzymatic degradation was studied using trypsin. Rheological studies revealed that hydrogels crosslinked with ELP dendrimers (HA_denELPs) degraded more slowly than those crosslinked with the otherwise equivalent linear ELPs (i.e., both molecules have the same number of pentamers and peripheral lysine residues). The origin of this phenomenon was evaluated using solution studies in which various dendritic and linear ELPs were treated with trypsin. Apart from the expected steric hindrances due to the dendritic topology, we identified the dual directionality of the peptide sequences (generated by a central branching lysine residue) and the likelihood of cleaving a productive crosslinking point as two additional contributors to the lesser degradability of HA_denELPs. Overall, these results highlight how the molecular design of crosslinker topology represents a novel strategy to tune the degradation rate of hydrogels.
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Ácido Hialurónico , Hidrogeles , Secuencia de Aminoácidos , Elastina , PéptidosRESUMEN
Polymer micelles, used extensively as vehicles in the delivery of active pharmaceutical ingredients, represent a versatile polymer architecture in drug delivery systems. We hypothesized that degree of crosslinking in the hydrophobic core of amphiphilic block copolymer micelles could be used to tune the rate of release of the biological signaling gas (gasotransmitter) hydrogen sulfide (H2S), a potential therapeutic. To test this hypothesis, we first synthesized amphiphilic block copolymers of the structure PEG-b-P(FBEA) (PEG = poly(ethylene glycol), FBEA = 2-(4-formylbenzoyloxy)ethyl acrylate). Using a modified arm-first approach, we then varied the crosslinking percentage in the core-forming block via addition of a 'O,O'-alkanediyl bis(hydroxylamine) crosslinking agent. We followed incorporation of the crosslinker by 1H NMR spectroscopy, monitoring the appearance of the oxime signal resulting from reaction of pendant aryl aldehydes on the block copolymer with hydroxylamines on the crosslinker, which revealed crosslinking percentages of 5, 10, and 15%. We then installed H2S-releasing S-aroylthiooxime (SATO) groups on the crosslinked polymers, yielding micelles with SATO units in their hydrophobic cores after self-assembly in water. H2S release studies in water, using cysteine (Cys) as a trigger to induce H2S release from the SATO groups in the micelle core, revealed increasing half-lives of H2S release, from 117 ± 6 min to 210 ± 30 min, with increasing crosslinking density in the micelle core. This result was consistent with our hypothesis, and we speculate that core crosslinking limits the rate of Cys diffusion into the micelle core, decreasing the release rate. This method for tuning the release of covalently linked small molecules through modulation of micelle core crosslinking density may extend beyond H2S to other drug delivery systems where precise control of release rate is needed.
RESUMEN
Overproduction of superoxide anion (O2.- ), the primary cellular reactive oxygen species (ROS), is implicated in various human diseases. To reduce cellular oxidative stress caused by overproduction of superoxide, we developed a compound that reacts with O2.- to release a persulfide (RSSH), a type of reactive sulfur species related to the gasotransmitter hydrogen sulfide (H2 S). Termed SOPD-NAC, this persulfide donor reacts specifically with O2.- , decomposing to generate N-acetyl cysteine (NAC) persulfide. To enhance persulfide delivery to cells, we conjugated the SOPD motif to a short, self-assembling peptide (Bz-CFFE-NH2 ) to make a superoxide-responsive, persulfide-donating peptide (SOPD-Pep). Both SOPD-NAC and SOPD-Pep delivered persulfides/H2 S to H9C2 cardiomyocytes and lowered ROS levels as confirmed by quantitative in vitro fluorescence imaging studies. Additional in vitro studies on RAW 264.7 macrophages showed that SOPD-Pep mitigated toxicity induced by phorbol 12-myristate 13-acetate (PMA) more effectively than SOPD-NAC and several control compounds, including common H2 S donors.
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Profármacos/farmacología , Sulfuros/farmacología , Superóxidos/antagonistas & inhibidores , Animales , Línea Celular , Sulfuro de Hidrógeno/química , Sulfuro de Hidrógeno/farmacología , Ratones , Estructura Molecular , Imagen Óptica , Estrés Oxidativo/efectos de los fármacos , Profármacos/síntesis química , Profármacos/química , Células RAW 264.7 , Ratas , Sulfuros/síntesis química , Sulfuros/química , Superóxidos/metabolismoRESUMEN
BACKGROUND: The continuum of pro- and anti-inflammatory response elicited by traumatic brain injury (TBI) is suggested to play a key role in the outcome of TBI; however, the underlying mechanisms remain ill -defined. METHODS: Here, we demonstrate that using bone marrow chimeric mice and systemic inhibition of EphA4 receptor shifts the pro-inflammatory milieu to pro-resolving following acute TBI. RESULTS: EphA4 expression is increased in the injured cortex as early as 2 h post-TBI and on CX3CR1gfp-positive cells in the peri-lesion. Systemic inhibition or genetic deletion of EphA4 significantly reduced cortical lesion volume and shifted the inflammatory profile of peripheral-derived immune cells to pro-resolving in the damaged cortex. These findings were consistent with in vitro studies showing EphA4 inhibition or deletion altered the inflammatory state of LPS-stimulated monocyte/macrophages towards anti-inflammatory. Phosphoarray analysis revealed that EphA4 may regulate pro-inflammatory gene expression by suppressing the mTOR, Akt, and NF-κB pathways. Our human metadata analysis further demonstrates increased EPHA4 and pro-inflammatory gene expression, which correlates with reduced AKT concurrent with increased brain injury severity in patients. CONCLUSIONS: Overall, these findings implicate EphA4 as a novel mediator of cortical tissue damage and neuroinflammation following TBI.
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Lesiones Traumáticas del Encéfalo/metabolismo , Corteza Cerebral/metabolismo , Encefalitis/metabolismo , Receptor EphA4/metabolismo , Animales , Barrera Hematoencefálica/metabolismo , Barrera Hematoencefálica/patología , Lesiones Traumáticas del Encéfalo/patología , Corteza Cerebral/patología , Modelos Animales de Enfermedad , Encefalitis/patología , Humanos , Masculino , Ratones , Ratones Transgénicos , Microglía/metabolismo , Microglía/patología , Receptor EphA4/genéticaRESUMEN
Volanesorsen (previously known as ISIS 304801) is a 20-nucleotide partially 2'-O-(2-methoxyethyl) (2'-MOE)-modified antisense oligonucleotide (ASO) gapmer, which was recently approved in the European Union as a novel, first-in-class treatment in the reduction of triglyceride levels in patients with familial chylomicronemia syndrome. We characterized the absorption, distribution, metabolism, and excretion characteristics of volanesorsen in mice, rats, monkeys, and humans, in either radiolabeled or nonradiolabeled studies. This also included the characterization of all of the observed ASO metabolite species excreted in urine. Volanesorsen is highly bound to plasma proteins that are similar in mice, monkeys, and humans. In all species, plasma concentrations declined in a multiphasic fashion, characterized by a relatively fast initial distribution phase and then a much slower terminal elimination phase following subcutaneous bolus administration. The plasma metabolite profiles of volanesorsen are similar across species, with volanesorsen as the major component. Various shortened oligonucleotide metabolites (5-19 nucleotides long) were identified in tissues in the multiple-dose mouse and monkey studies, but fewer in the [3H]-volanesorsen rat study, likely due to a lower accumulation of metabolites following a single dose in rats. In urine, all metabolites identified in tissues were observed, consistent with both endo- and exonuclease-mediated metabolism and urinary excretion being the major elimination pathway for volanesorsen and its metabolites. SIGNIFICANCE STATEMENT: We characterized the absorption, distribution, metabolism, and excretion (ADME) of volanesorsen, a partially 2'-MOE-modified antisense oligonucleotide, from mouse to man utilizing novel extraction and quantitation techniques in samples collected from preclinical toxicology studies, a 3H rat ADME study, and a phase 1 clinical trial.
Asunto(s)
Apolipoproteína C-III/antagonistas & inhibidores , Proteínas Sanguíneas/metabolismo , Oligonucleótidos/farmacocinética , Adulto , Animales , Apolipoproteína C-III/genética , Apolipoproteína C-III/metabolismo , Evaluación Preclínica de Medicamentos/métodos , Femenino , Voluntarios Sanos , Humanos , Hiperlipoproteinemia Tipo I/sangre , Hiperlipoproteinemia Tipo I/tratamiento farmacológico , Hiperlipoproteinemia Tipo I/genética , Inyecciones Subcutáneas , Macaca fascicularis , Masculino , Tasa de Depuración Metabólica , Ratones , Persona de Mediana Edad , Mutación , Oligonucleótidos/administración & dosificación , Ratas , Eliminación Renal , Especificidad de la Especie , Distribución Tisular , Triglicéridos/sangre , Triglicéridos/metabolismoRESUMEN
H2S is a gasotransmitter with several physiological roles, but its reactivity and short half-life in biological media make its controlled delivery difficult. For biological applications of the gas, hydrogels have the potential to deliver H2S with several advantages over other donor systems, including localized delivery, controlled release rates, biodegradation, and variable mechanical properties. In this study, we designed and evaluated peptide-based H2S-releasing hydrogels with controllable H2S delivery. The hydrogels were prepared from short, self-assembling aromatic peptide amphiphiles (APAs), functionalized on their N-terminus with S-aroylthiooximes (SATOs), which release H2S in response to a thiol trigger. The APAs were studied both in solution and in gel forms, with gelation initiated by addition of CaCl2. Various substituents were included on the SATO component of the APAs in order to evaluate their effects on self-assembled morphology and H2S release rate in both the solution and gel phases. Transmission electron microscopy (TEM) images confirmed that all H2S-releasing APAs self-assembled into nanofibers above a critical aggregation concentration (CAC) of â¼0.5 mg/mL. Below the CAC, substituents on the SATO group affected H2S release rates predictably in line with electronic effects (Hammett σ values) according to a linear free energy relationship. Above the CAC, circular dichroism, infrared, and fluorescence spectroscopies demonstrated that substituents influenced the self-assembled structures by affecting hydrogen bonding (ß-sheet formation) and π-π stacking. At these concentrations, electronic control over release rates diminished, both in solution and in the gel form. Rather, the release rate depended primarily on the degree of organization in the ß-sheets and the amount of π-π stacking. This supramolecular control over release rate may enable the evaluation of H2S-releasing hydrogels with different release rates in biological applications.