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1.
Sci Rep ; 11(1): 5749, 2021 03 11.
Artigo em Inglês | MEDLINE | ID: mdl-33707480

RESUMO

Reactive oxygen species (ROS) are implicated in triggering cell signalling events and pathways to promote and maintain tumorigenicity. Chemotherapy and radiation can induce ROS to elicit cell death allows for targeting ROS pathways for effective anti-cancer therapeutics. Coenzyme Q10 is a critical cofactor in the electron transport chain with complex biological functions that extend beyond mitochondrial respiration. This study demonstrates that delivery of oxidized Coenzyme Q10 (ubidecarenone) to increase mitochondrial Q-pool is associated with an increase in ROS generation, effectuating anti-cancer effects in a pancreatic cancer model. Consequent activation of cell death was observed in vitro in pancreatic cancer cells, and both human patient-derived organoids and tumour xenografts. The study is a first to demonstrate the effectiveness of oxidized ubidecarenone in targeting mitochondrial function resulting in an anti-cancer effect. Furthermore, these findings support the clinical development of proprietary formulation, BPM31510, for treatment of cancers with high ROS burden with potential sensitivity to ubidecarenone.


Assuntos
Apoptose , Mitocôndrias/metabolismo , Neoplasias Pancreáticas/patologia , Espécies Reativas de Oxigênio/metabolismo , Ubiquinona/análogos & derivados , Animais , Linhagem Celular Tumoral , Proliferação de Células , Respiração Celular , Sobrevivência Celular , Complexo II de Transporte de Elétrons/metabolismo , Glicerol-3-Fosfato Desidrogenase (NAD+) , Humanos , Potencial da Membrana Mitocondrial , Camundongos Nus , Organoides/patologia , Estresse Oxidativo , Consumo de Oxigênio , Neoplasias Pancreáticas/metabolismo , Especificidade por Substrato , Ubiquinona/metabolismo
2.
PLoS Pathog ; 16(5): e1008539, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32459815

RESUMO

NAD, a key co-enzyme required for cell metabolism, is synthesized via two pathways in most organisms. Since schistosomes apparently lack enzymes required for de novo NAD biosynthesis, we evaluated whether these parasites, which infect >200 million people worldwide, maintain NAD homeostasis via the NAD salvage biosynthetic pathway. We found that intracellular NAD levels decline in schistosomes treated with drugs that block production of nicotinamide or nicotinamide mononucleotide-known NAD precursors in the non-deamidating salvage pathway. Moreover, in vitro inhibition of the NAD salvage pathway in schistosomes impaired egg production, disrupted the outer membranes of both immature and mature parasites and caused loss of mobility and death. Inhibiting the NAD salvage pathway in schistosome-infected mice significantly decreased NAD levels in adult parasites, which correlated with reduced egg production, fewer liver granulomas and parasite death. Thus, schistosomes, unlike their mammalian hosts, appear limited to one metabolic pathway to maintain NAD-dependent metabolic processes.


Assuntos
Interações Hospedeiro-Parasita/fisiologia , NAD/metabolismo , Schistosoma mansoni/fisiologia , Esquistossomose mansoni/metabolismo , Animais , Feminino , Camundongos , Reprodução/fisiologia , Esquistossomose mansoni/patologia
3.
Ann Neurol ; 78(1): 88-103, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25893674

RESUMO

OBJECTIVE: Alzheimer's disease (AD)-associated dementia is due to tissue damage caused by amyloid ß (Aß) deposition within the brain and by accompanying neuroinflammation. The nicotinamide adenine dinucleotide (NAD) glycohydrolase CD38, which is expressed by neurons, astrocytes, and microglial cells, regulates inflammatory and repair processes in the brain and other tissues by degrading NAD and repressing the activity of other NAD-consuming enzymes and by producing NAD-derived metabolites that regulate calcium signaling and migration of inflammatory cells. Given the role of CD38 in neuroinflammation and repair, we examined the effect of CD38 deletion on AD pathology. METHODS: We crossed APPswePS1ΔE9 (APP.PS) mice with Cd38(-) (/) (-) mice to generate AD-prone CD38-deficient animals (APP.PS.Cd38(-) (/) (-) ) and examined AD-related phenotypes in both groups. RESULTS: APP.PS.Cd38(-) (/) (-) mice exhibited significant reductions in Aß plaque load and soluble Aß levels compared to APP.PS mice, and this correlated with improved spatial learning. Although CD38 deficiency resulted in decreased microglia/macrophage (MM) accumulation, the transcription profile of the Cd38(-) (/) (-) and Cd38(+/) (+) MM was similar, suggesting that the decreased Aß burden in APP.PS.Cd38(-) (/) (-) mice was not due to alterations in MM activation/function. Instead, APP.PS.Cd38(-) (/) (-) neuronal cultures secreted less Aß and this reduction was mimicked when APP.PS neuronal cultures were treated with inhibitors that blocked CD38 enzyme activity or the signaling pathways controlled by CD38-derived metabolites. Furthermore, ß- and γ-secretase activity was decreased in APP.PS.Cd38(-) (/) (-) mice, which correlated with decreased Aß production. INTERPRETATION: CD38 regulates AD pathology in the APP.PS model of AD, suggesting that CD38 may be a novel target for AD treatment.


Assuntos
ADP-Ribosil Ciclase 1/genética , Doença de Alzheimer/genética , Secretases da Proteína Precursora do Amiloide/metabolismo , Peptídeos beta-Amiloides/metabolismo , Comportamento Animal , Encéfalo/patologia , Glicoproteínas de Membrana/genética , Placa Amiloide/patologia , RNA Mensageiro/metabolismo , Precursor de Proteína beta-Amiloide/genética , Animais , Encéfalo/metabolismo , Movimento Celular , Células Cultivadas , Modelos Animais de Doenças , Macrófagos/metabolismo , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Microglia/metabolismo , Placa Amiloide/metabolismo , Aprendizagem Espacial , Transcriptoma
4.
J Tissue Eng ; 5: 2041731414528736, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24812579

RESUMO

Advances in allograft processing have opened new horizons for clinical adaptation of flexor tendon allografts as delivery scaffolds for antifibrotic therapeutics. Recombinant adeno-associated-virus (rAAV) gene delivery of the growth and differentiation factor 5 (GDF-5) has been previously associated with antifibrotic effects in a mouse model of flexor tendoplasty. In this study, we compared the effects of loading freeze-dried allografts with different doses of GDF-5 protein or rAAV-Gdf5 on flexor tendon healing and adhesions. We first optimized the protein and viral loading parameters using reverse transcription polymerase chain reaction (RT-PCR), enzyme-linked immunosorbent assay (ELISA), and in vivo bioluminescent imaging. We then reconstructed flexor digitorum longus (FDL) tendons of the mouse hindlimb with allografts loaded with low and high doses of recombinant GDF-5 protein and rAAV-Gdf5 and evaluated joint flexion and biomechanical properties of the reconstructed tendon. In vitro optimization studies determined that both the loading time and concentration of the growth factor and viral vector had dose-dependent effects on their retention on the freeze-dried allograft. In vivo data suggest that protein and gene delivery of GDF-5 had equivalent effects on improving joint flexion function, in the range of doses used. Within the doses tested, the lower doses of GDF-5 had more potent effects on suppressing adhesions without adversely affecting the strength of the repair. These findings indicate equivalent antifibrotic effects of Gdf5 gene and protein delivery, but suggest that localized delivery of this potent factor should also carefully consider the dosage used to eliminate untoward effects, regardless of the delivery mode.

5.
Tissue Eng Part A ; 17(3-4): 389-98, 2011 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-20807012

RESUMO

To investigate the efficacy of endocrine parathyroid hormone treatment on tissue-engineered bone regeneration, massive femoral defects in C57Bl/6 mice were reconstructed with either 100:0 or 85:15 poly-lactic acid (PLA)/beta-tricalcium phosphate (ß-TCP) scaffolds (hereafter PLA or PLA/ßTCP, respectively), which were fabricated with low porosity (<30%) to improve their structural rigidity. Experimental mice were treated starting at 1 week postop with daily subcutaneous injections of 40 µg/kg teriparatide until sacrifice at 9 weeks, whereas control mice underwent the same procedure but were injected with sterile saline. Bone regeneration was assessed longitudinally using planar X-ray and quantitative microcomputed tomography, and the reconstructed femurs were evaluated at 9 weeks either histologically or biomechanically to determine their torsional strength and rigidity. Teriparatide treatment increased bone volume and bone mineral content significantly at 6 weeks and led to enhanced trabeculated bone callus formation that appeared to surround and integrate with the scaffold, thereby establishing union by bridging bone regeneration across the segmental defect in 30% of the reconstructed femurs, regardless of the scaffold type. However, the bone volume and mineral content in the PLA reconstructed femurs treated with teriparatide was reduced at 9 weeks to control levels, but remained significantly increased in the PLA/ßTCP scaffolds. Further, bridged teriparatide-treated femurs demonstrated a prototypical brittle bone torsion behavior, and were significantly stronger and stiffer than control specimens or treated specimens that failed to form bridging bone union. Taken together, these observations suggest that intermittent, systemic parathyroid hormone treatment can enhance bone regeneration in scaffold-reconstructed femoral defects, which can be further enhanced by mineralized (ßTCP) particles within the scaffold.


Assuntos
Substitutos Ósseos/uso terapêutico , Fosfatos de Cálcio/uso terapêutico , Fraturas do Fêmur/terapia , Teriparatida/administração & dosagem , Alicerces Teciduais , Animais , Terapia Combinada , Feminino , Fraturas do Fêmur/patologia , Camundongos , Camundongos Endogâmicos BALB C , Resultado do Tratamento
6.
Mol Ther ; 16(3): 466-73, 2008 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-18180771

RESUMO

Tendon reconstruction using grafts often results in adhesions that limit joint flexion. These adhesions are precipitated by inflammation, fibrosis, and the paucity of tendon differentiation signals during healing. In order to study this problem, we developed a mouse model in which the flexor digitorum longus (FDL) tendon is reconstructed using a live autograft or a freeze-dried allograft, and identified growth and differentiation factor 5 (Gdf5) as a therapeutic target. In this study we have investigated the potential of rAAV-Gdf5 -loaded freeze-dried tendon allografts as "therapeutically endowed" tissue-engineering scaffolds to reduce adhesions. In reporter gene studies we have demonstrated that recombinant adeno-associated virus (rAAV)-loaded tendon allografts mediate efficient transduction of adjacent soft tissues, with expression peaking at 7 days. We have also demonstrated that the rAAV-Gdf5 vector significantly accelerates wound healing in an in vitro fibroblast scratch model and, when loaded onto freeze-dried FDL tendon allografts, improves the metatarsophalangeal (MTP) joint flexion to a significantly greater extent than the rAAV-lacZ controls do. Collectively, our data demonstrate the feasibility and efficacy of therapeutic tendon allograft processing as a novel paradigm in tissue engineering in order to address difficult clinical problems such as tendon adhesions.


Assuntos
Proteínas Morfogenéticas Ósseas/fisiologia , Artropatias/terapia , Tendões/transplante , Engenharia Tecidual/métodos , Animais , Proteínas Morfogenéticas Ósseas/genética , Dependovirus/genética , Liofilização , Terapia Genética/métodos , Vetores Genéticos/genética , Fator 5 de Diferenciação de Crescimento , Imuno-Histoquímica , Artropatias/genética , Cinética , Camundongos , Camundongos Endogâmicos C57BL , Reação em Cadeia da Polimerase , Alicerces Teciduais , Transdução Genética , Transplante Homólogo , Cicatrização/genética , Cicatrização/fisiologia
7.
J Orthop Res ; 26(6): 824-33, 2008 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-18186128

RESUMO

Reconstruction of flexor tendons often results in adhesions that compromise joint flexion. Little is known about the factors involved in the formation of flexor tendon graft adhesions. In this study, we developed and characterized a novel mouse model of flexor digitorum longus (FDL) tendon reconstruction with live autografts or reconstituted freeze-dried allografts. Grafted tendons were evaluated at multiple time points up to 84 days post-reconstruction. To assess the flexion range of the metatarsophalangeal joint, we developed a quantitative outcome measure proportional to the resistance to tendon gliding due to adhesions, which we termed the Gliding Coefficient. At 14 days post-grafting, the Gliding Coefficient was 29- and 26-fold greater than normal FDL tendon for both autografts and allografts, respectively (p < 0.001), and subsequently doubled for 28-day autografts. Interestingly, there were no significant differences in maximum tensile force or stiffness between live autograft and freeze-dried allograft repairs over time. Histologically, autograft healing was characterized by extensive remodeling and exuberant scarring around both the ends and the body of the graft, whereas allograft scarring was abundant only near the graft-host junctions. Gene expression of GDF-5 and VEGF were significantly increased in 28-day autografts compared to allografts and to normal tendons. These results suggest that the biomechanical advantages for tendon reconstruction using live autografts over devitalized allografts are minimal. This mouse model can be useful in elucidating the molecular mechanisms in tendon repair and can aid in preliminary screening of molecular treatments of flexor tendon adhesions.


Assuntos
Tendão do Calcâneo/fisiologia , Tendão do Calcâneo/transplante , Procedimentos Ortopédicos/métodos , Procedimentos de Cirurgia Plástica/métodos , Aderências Teciduais/prevenção & controle , Tendão do Calcâneo/patologia , Actinas/genética , Animais , Fenômenos Biomecânicos , Proteínas Morfogenéticas Ósseas/genética , Liofilização , Expressão Gênica , Fator 5 de Diferenciação de Crescimento , Articulação Metatarsofalângica/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Modelos Animais , Amplitude de Movimento Articular , Aderências Teciduais/patologia , Aderências Teciduais/fisiopatologia , Fator de Crescimento Transformador beta1/genética , Transplante Autólogo , Transplante Homólogo , Fator A de Crescimento do Endotélio Vascular/genética
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