RESUMO
Background: The outer mitochondrial Rho GTPase 1, MIRO1, mediates mitochondrial motility within cells, but implications for vascular smooth muscle cell (VSMC) physiology and its roles invascular diseases, such as neointima formation following vascular injury are widely unknown. Methods: An in vivo model of selective Miro1 deletion in VSMCs was generated, and the animals were subjected to carotid artery ligation. The molecular mechanisms relevant to VSMC proliferation were then explored in explanted VSMCs by imaging mitochondrial positioning and cristae structure and assessing the effects on ATP production, metabolic function and interactions with components of the electron transport chain (ETC). Results: MIRO1 was robustly expressed in VSMCs within human atherosclerotic plaques and promoted VSMC proliferation and neointima formation in mice by blocking cell-cycle progression at G1/S, mitochondrial positioning, and PDGF-induced ATP production and respiration; overexpression of a MIRO1 mutant lacking the EF hands that are required for mitochondrial mobility did not fully rescue these effects. At the ultrastructural level, Miro1 deletion distorted the mitochondrial cristae and reduced the formation of super complexes and the activity of ETC complex I. Conclusions: Mitochondrial motility is essential for VSMC proliferation and relies on MIRO1. The EF-hands of MIRO1 regulate the intracellular positioning of mitochondria. Additionally, the absence of MIRO1 leads to distorted mitochondrial cristae and reduced ATP generation. Our findings demonstrate that motility is linked to mitochondrial ATP production. We elucidated two unrecognized mechanisms through which MIRO1 influences cell proliferation by modulating mitochondria: first, by managing mitochondrial placement via Ca2+-dependent EF hands, and second, by affecting cristae structure and ATP synthesis.
Assuntos
Nefropatias Diabéticas , Dineínas , Animais , Humanos , Proteínas Quinases Ativadas por AMP/metabolismo , Proteínas Quinases Ativadas por AMP/genética , Nefropatias Diabéticas/metabolismo , Nefropatias Diabéticas/genética , Dineínas/metabolismo , Dineínas/genética , Metabolismo Energético/efeitos dos fármacos , Fator de Transcrição Sp1RESUMO
Changes in the anterior segment of the eye due to type 2 diabetes mellitus (T2DM) are not well-characterized, in part due to the lack of a reliable animal model. This study evaluated changes in the anterior segment, including crystalline lens health, corneal endothelial cell density, aqueous humor metabolites, and ciliary body vasculature, in a rat model of T2DM compared with human eyes. Male Sprague-Dawley rats were fed a high-fat diet (45% fat) or normal diet, and rats fed the high-fat diet were injected with streptozotocin intraperitoneally to generate a model of T2DM. Cataract formation and corneal endothelial cell density were assessed using microscopic analysis. Diabetes-related rat aqueous humor alterations were assessed using metabolomics screening. Transmission electron microscopy was used to assess qualitative ultrastructural changes ciliary process microvessels at the site of aqueous formation in the eyes of diabetic rats and humans. Eyes from the diabetic rats demonstrated cataracts, lower corneal endothelial cell densities, altered aqueous metabolites, and ciliary body ultrastructural changes, including vascular endothelial cell activation, pericyte degeneration, perivascular edema, and basement membrane reduplication. These findings recapitulated diabetic changes in human eyes. These results support the use of this model for studying ocular manifestations of T2DM and support a hypothesis postulating blood-aqueous barrier breakdown and vascular leakage at the ciliary body as a mechanism for diabetic anterior segment pathology.
Assuntos
Diabetes Mellitus Experimental , Diabetes Mellitus Tipo 2 , Ratos Sprague-Dawley , Animais , Diabetes Mellitus Tipo 2/patologia , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/complicações , Masculino , Ratos , Humanos , Diabetes Mellitus Experimental/patologia , Diabetes Mellitus Experimental/complicações , Modelos Animais de Doenças , Segmento Anterior do Olho/patologia , Humor Aquoso/metabolismo , Catarata/patologia , Catarata/metabolismo , Cristalino/patologia , Cristalino/metabolismo , Cristalino/ultraestrutura , Corpo Ciliar/patologia , Corpo Ciliar/metabolismo , Dieta Hiperlipídica/efeitos adversosAssuntos
Fenômenos Bioquímicos , Diabetes Mellitus , Humanos , Dineínas/metabolismo , Transporte ProteicoRESUMO
Insulin and insulin-like growth factor 1 (IGF1) signaling is transduced by insulin receptor substrate 1 (IRS1) and IRS2. To elucidate physiological and redundant roles of insulin and IGF1 signaling in adult hearts, we generated mice with inducible cardiomyocyte-specific deletion of insulin and IGF1 receptors or IRS1 and IRS2. Both models developed dilated cardiomyopathy, and most mice died by 8 weeks post-gene deletion. Heart failure was characterized by cardiomyocyte loss and disarray, increased proapoptotic signaling, and increased autophagy. Suppression of autophagy by activating mTOR signaling did not prevent heart failure. Transcriptional profiling revealed reduced serum response factor (SRF) transcriptional activity and decreased mRNA levels of genes encoding sarcomere and gap junction proteins as early as 3 days post-gene deletion, in concert with ultrastructural evidence of sarcomere disruption and intercalated discs within 1 week after gene deletion. These data confirm conserved roles for constitutive insulin and IGF1 signaling in suppressing autophagic and apoptotic signaling in the adult heart. The present study also identifies an unexpected role for insulin and IGF1 signaling in regulating an SRF-mediated transcriptional program, which maintains expression of genes encoding proteins that support sarcomere integrity in the adult heart, reduction of which results in rapid development of heart failure.
Assuntos
Insuficiência Cardíaca , Fator de Crescimento Insulin-Like I , Camundongos , Animais , Proteínas Substratos do Receptor de Insulina/metabolismo , Fator de Crescimento Insulin-Like I/genética , Insulina/metabolismo , Fator de Resposta Sérica/metabolismo , Sarcômeros/metabolismo , Miócitos Cardíacos/metabolismo , Insuficiência Cardíaca/metabolismo , Serina-Treonina Quinases TOR/metabolismo , RNA Mensageiro/metabolismo , Conexinas/metabolismoRESUMO
Studies of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-infected patients and experimentally infected animals indicate a critical role for augmented expression of proinflammatory chemokines and cytokines in severe disease. Here, we demonstrate that SARS-CoV-2 infection of human monocyte-derived macrophages (MDMs) and monocyte-derived dendritic cells was abortive, but induced the production of multiple antiviral and proinflammatory cytokines (interferon-α, interferon-ß, tumor necrosis factor, and interleukins 1ß, 6, and 10) and a chemokine (CXCL10). Despite the lack of efficient replication in MDMs, SARS-CoV-2 induced profound interferon-mediated cell death of host cells. Macrophage activation and death were not enhanced by exposure to low levels of convalescent plasma, suggesting that antibody-dependent enhancement of infection does not contribute to cell death. Together, these results indicate that infection of macrophages and dendritic cells potentially plays a major role in coronavirus disease 2019 pathogenesis, even in the absence of productive infection.
Assuntos
COVID-19/terapia , Células Dendríticas/virologia , Macrófagos/virologia , SARS-CoV-2/imunologia , COVID-19/imunologia , Morte Celular , Quimiocinas/genética , Quimiocinas/metabolismo , Citocinas/genética , Citocinas/metabolismo , Células Dendríticas/imunologia , Células Dendríticas/ultraestrutura , Humanos , Imunização Passiva , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/virologia , Macrófagos/imunologia , Macrófagos/ultraestrutura , Microscopia Eletrônica de Transmissão , RNA Mensageiro/metabolismo , RNA Viral/metabolismo , Soroterapia para COVID-19RESUMO
OBJECTIVE: The main objective of this study is to define the mechanisms by which mitochondria control vascular smooth muscle cell (VSMC) migration and impact neointimal hyperplasia. APPROACH AND RESULTS: The multifunctional CaMKII (Ca2+/calmodulin-dependent kinase II) in the mitochondrial matrix of VSMC drove a feed-forward circuit with the mitochondrial Ca2+ uniporter (MCU) to promote matrix Ca2+ influx. MCU was necessary for the activation of mitochondrial CaMKII (mtCaMKII), whereas mtCaMKII phosphorylated MCU at the regulatory site S92 that promotes Ca2+ entry. mtCaMKII was necessary and sufficient for platelet-derived growth factor-induced mitochondrial Ca2+ uptake. This effect was dependent on MCU. mtCaMKII and MCU inhibition abrogated VSMC migration and mitochondrial translocation to the leading edge. Overexpression of wild-type MCU, but not MCU S92A, mutant in MCU-/- VSMC rescued migration and mitochondrial mobility. Inhibition of microtubule, but not of actin assembly, blocked mitochondrial mobility. The outer mitochondrial membrane GTPase Miro-1 promotes mitochondrial mobility via microtubule transport but arrests it in subcellular domains of high Ca2+ concentrations. In Miro-1-/- VSMC, mitochondrial mobility and VSMC migration were abolished, and overexpression of mtCaMKII or a CaMKII inhibitory peptide in mitochondria (mtCaMKIIN) had no effect. Consistently, inhibition of mtCaMKII increased and prolonged cytosolic Ca2+ transients. mtCaMKII inhibition diminished phosphorylation of focal adhesion kinase and myosin light chain, leading to reduced focal adhesion turnover and cytoskeletal remodeling. In a transgenic model of selective mitochondrial CaMKII inhibition in VSMC, neointimal hyperplasia was significantly reduced after vascular injury. CONCLUSIONS: These findings identify mitochondrial CaMKII as a key regulator of mitochondrial Ca2+ uptake via MCU, thereby controlling mitochondrial translocation and VSMC migration after vascular injury.
Assuntos
Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Cálcio/metabolismo , Lesões das Artérias Carótidas/enzimologia , Movimento Celular , Mitocôndrias Musculares/enzimologia , Músculo Liso Vascular/enzimologia , Miócitos de Músculo Liso/enzimologia , Neointima , Animais , Canais de Cálcio/genética , Canais de Cálcio/metabolismo , Sinalização do Cálcio , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Lesões das Artérias Carótidas/genética , Lesões das Artérias Carótidas/patologia , Células Cultivadas , Modelos Animais de Doenças , Hiperplasia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mitocôndrias Musculares/patologia , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/patologia , Proteínas rho de Ligação ao GTP/genética , Proteínas rho de Ligação ao GTP/metabolismoRESUMO
The multifunctional Ca2+/calmodulin-dependent protein kinase II (CaMKII) is a serine/threonine kinase important in transducing intracellular Ca2+ signals. While in vitro data regarding the role of CaMKII in the regulation of endothelial nitric oxide synthase (eNOS) are contradictory, its role in endothelial function in vivo remains unknown. Using two novel transgenic models to express CaMKII inhibitor peptides selectively in endothelium, we examined the effect of CaMKII on eNOS activation, NO production, vasomotor tone and blood pressure. Under baseline conditions, CaMKII activation was low in the aortic wall. Consistently, systolic and diastolic blood pressure, heart rate and plasma NO levels were unaltered by endothelial CaMKII inhibition. Moreover, endothelial CaMKII inhibition had no significant effect on NO-dependent vasodilation. These results were confirmed in studies of aortic rings transduced with adenovirus expressing a CaMKII inhibitor peptide. In cultured endothelial cells, bradykinin treatment produced the anticipated rapid influx of Ca2+ and transient CaMKII and eNOS activation, whereas CaMKII inhibition blocked eNOS phosphorylation on Ser-1179 and dephosphorylation at Thr-497. Ca2+/CaM binding to eNOS and resultant NO production in vitro were decreased under CaMKII inhibition. Our results demonstrate that CaMKII plays an important role in transient bradykinin-driven eNOS activation in vitro, but does not regulate NO production, vasorelaxation or blood pressure in vivo under baseline conditions.
Assuntos
Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Óxido Nítrico/metabolismo , Animais , Linhagem Celular , Humanos , FosforilaçãoRESUMO
High-dose chemotherapies to treat multiple myeloma (MM) can be life-threatening due to toxicities to normal cells and there is a need to target only tumor cells and/or lower standard drug dosage without losing efficacy. We show that pharmacologically-dosed ascorbic acid (PAA), in the presence of iron, leads to the formation of highly reactive oxygen species (ROS) resulting in cell death. PAA selectively kills CD138+ MM tumor cells derived from MM and smoldering MM (SMM) but not from monoclonal gammopathy undetermined significance (MGUS) patients. PAA alone or in combination with melphalan inhibits tumor formation in MM xenograft mice. This study shows PAA efficacy on primary cancer cells and cell lines in vitro and in vivo.
Assuntos
Apoptose/efeitos dos fármacos , Ácido Ascórbico/farmacologia , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Ácido Ascórbico/química , Ácido Ascórbico/uso terapêutico , Proteínas de Ligação ao Cálcio , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/metabolismo , Quimioterapia Combinada , Humanos , Ferro/química , Melfalan/uso terapêutico , Camundongos , Camundongos Endogâmicos NOD , Proteínas dos Microfilamentos , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/metabolismo , Mieloma Múltiplo/patologia , Espécies Reativas de Oxigênio/metabolismo , Sindecana-1/metabolismo , Transplante HeterólogoRESUMO
The calcium and calmodulin-dependent kinase II (CaMKII) translates increases in intracellular Ca(2+) into downstream signaling events. Its function in pulmonary pathologies remains largely unknown. CaMKII is a well-known mediator of apoptosis and regulator of endoplasmic reticulum (ER) Ca(2+). ER stress and apoptosis of type II pneumocytes lead to aberrant tissue repair and progressive collagen deposition in pulmonary fibrosis. Thus we hypothesized that CaMKII inhibition alleviates fibrosis in response to bleomycin by attenuating apoptosis and ER stress of type II pneumocytes. We first established that CaMKII was strongly expressed in the distal respiratory epithelium, in particular in surfactant protein-C-positive type II pneumocytes, and activated after bleomycin instillation. We generated a novel transgenic model of inducible expression of the CaMKII inhibitor peptide AC3-I limited to type II pneumocytes (Tg SPC-AC3-I). Tg SPC-AC3-I mice were protected from development of pulmonary fibrosis after bleomycin exposure compared with wild-type mice. CaMKII inhibition also provided protection from apoptosis in type II pneumocytes in vitro and in vivo. Moreover, intracellular Ca(2+) levels and ER stress were increased by bleomycin and significantly blunted with CaMKII inhibition in vitro. These data demonstrate that CaMKII inhibition prevents type II pneumocyte apoptosis and development of pulmonary fibrosis in response to bleomycin. CaMKII inhibition may therefore be a promising approach to prevent or ameliorate the progression of pulmonary fibrosis.
Assuntos
Células Epiteliais Alveolares/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Bleomicina/farmacologia , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/antagonistas & inibidores , Cálcio/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Fibrose Pulmonar/tratamento farmacológico , Células Epiteliais Alveolares/metabolismo , Animais , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Estresse do Retículo Endoplasmático/fisiologia , Camundongos Transgênicos , Fibrose Pulmonar/metabolismo , Fibrose Pulmonar/patologiaRESUMO
There exists a dire need for improved therapeutics to achieve predictable bone regeneration. Gene therapy using non-viral vectors that are safe and efficient at transfecting target cells is a promising approach to overcoming the drawbacks of protein delivery of growth factors. Here, we investigated the transfection efficiency, cytotoxicity, osteogenic potential and in vivo bone regenerative capacity of chemically modified ribonucleic acid (cmRNA) (encoding BMP-2) complexed with polyethylenimine (PEI) and made comparisons with PEI complexed with conventional plasmid DNA (encoding BMP-2). The polyplexes were fabricated at an amine (N) to phosphate (P) ratio of 10 and characterized for transfection efficiency using human bone marrow stromal cells (BMSCs). The osteogenic potential of BMSCs treated with these polyplexes was validated by determining the expression of bone-specific genes, osteocalcin and alkaline phosphatase as well as through the detection of bone matrix deposition. Using a calvarial bone defect model in rats, it was shown that PEI-cmRNA (encoding BMP-2)-activated matrices promoted significantly enhanced bone regeneration compared to PEI-plasmid DNA (BMP-2)-activated matrices. Our proof of concept study suggests that scaffolds loaded with non-viral vectors harboring cmRNA encoding osteogenic proteins may be a powerful tool for stimulating bone regeneration with significant potential for clinical translation.
Assuntos
Proteína Morfogenética Óssea 2/genética , Regeneração Óssea , Polietilenoimina/química , RNA/administração & dosagem , RNA/química , Fosfatase Alcalina/genética , Animais , Células da Medula Óssea/citologia , Sobrevivência Celular , Traumatismos Craniocerebrais/terapia , DNA/administração & dosagem , DNA/química , Terapia Genética , Humanos , Masculino , Camundongos Endogâmicos BALB C , Osteocalcina/genética , Plasmídeos , RNA/farmacologia , RNA/uso terapêutico , Ratos Endogâmicos F344 , Células Estromais/metabolismoRESUMO
Compartmentalization and polarized protein trafficking are essential for many cellular functions. The photoreceptor outer segment (OS) is a sensory compartment specialized for phototransduction, and it shares many features with primary cilia. As expected, mutations disrupting protein trafficking to cilia often disrupt protein trafficking to the OS and cause photoreceptor degeneration. Bardet-Biedl syndrome (BBS) is one of the ciliopathies associated with defective ciliary trafficking and photoreceptor degeneration. However, precise roles of BBS proteins in photoreceptor cells and the underlying mechanisms of photoreceptor degeneration in BBS are not well understood. Here, we show that accumulation of non-OS proteins in the OS underlies photoreceptor degeneration in BBS. Using a newly developed BBS mouse model [Leucine zipper transcription factor-like 1 (Lztfl1)/Bbs17 mutant], isolated OSs, and quantitative proteomics, we determined 138 proteins that are enriched more than threefold in BBS mutant OS. In contrast, only eight proteins showed a more than threefold reduction. We found striking accumulation of Stx3 and Stxbp1/Munc18-1 and loss of polarized localization of Prom1 within the Lztfl1 and Bbs1 mutant OS. Ultrastructural analysis revealed that large vesicles are formed in the BBS OS, disrupting the lamellar structure of the OS. Our findings suggest that accumulation (and consequent sequestration) of non-OS proteins in the OS is likely the primary cause of photoreceptor degeneration in BBS. Our data also suggest that a major function of BBS proteins in photoreceptors is to transport proteins from the OS to the cell body or to prevent entry of non-OS proteins into the OS.
Assuntos
Síndrome de Bardet-Biedl/metabolismo , Síndrome de Bardet-Biedl/patologia , Proteínas do Olho/metabolismo , Degeneração Retiniana/metabolismo , Degeneração Retiniana/patologia , Segmento Externo das Células Fotorreceptoras da Retina/metabolismo , Segmento Externo das Células Fotorreceptoras da Retina/patologia , Animais , Especificidade de Anticorpos , Western Blotting , Separação Celular , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Obesidade/complicações , Obesidade/patologia , Proteômica , Reprodutibilidade dos Testes , Degeneração Retiniana/complicações , Degeneração Retiniana/fisiopatologia , Segmento Externo das Células Fotorreceptoras da Retina/ultraestrutura , Fatores de Transcrição/metabolismo , UltracentrifugaçãoRESUMO
Alcohol is the most commonly abused drug worldwide, and chronic alcohol consumption is a major etiological factor in the development of multiple pathological sequelae, including alcoholic cardiomyopathy and hepatic cirrhosis. Here, we identify regulator of G protein signaling 6 (RGS6) as a critical regulator of both alcohol-seeking behaviors and the associated cardiac and hepatic morbidities through two mechanistically divergent signaling actions. RGS6(-/-) mice consume less alcohol when given free access and are less susceptible to alcohol-induced reward and withdrawal. Antagonism of GABA(B) receptors or dopamine D2 receptors partially reversed the reduction in alcohol consumption in RGS6(-/-) animals. Strikingly, dopamine transporter inhibition completely restored alcohol seeking in mice lacking RGS6. RGS6 deficiency was associated with alterations in the expression of genes controlling dopamine (DA) homeostasis and a reduction in DA levels in the striatum. Taken together, these data implicate RGS6 as an essential regulator of DA bioavailability. RGS6 deficiency also provided dramatic protection against cardiac hypertrophy and fibrosis, hepatic steatosis, and gastrointestinal barrier dysfunction and endotoxemia when mice were forced to consume alcohol. Although RGS proteins canonically function as G-protein regulators, RGS6-dependent, alcohol-mediated toxicity in the heart, liver, and gastrointestinal tract involves the ability of RGS6 to promote reactive oxygen species-dependent apoptosis, an action independent of its G-protein regulatory capacity. We propose that inhibition of RGS6 might represent a viable means to reduce alcohol cravings and withdrawal in human patients, while simultaneously protecting the heart and liver from further damage upon relapse.
Assuntos
Consumo de Bebidas Alcoólicas , Comportamento Animal , Proteínas RGS/fisiologia , Recompensa , Animais , Apoptose/fisiologia , Cardiomiopatias/etiologia , Condicionamento Operante , Camundongos , Camundongos Knockout , Proteínas RGS/genéticaRESUMO
Type 1 diabetes (T1D) is caused by autoimmune disease that leads to the destruction of pancreatic ß-cells. Transplantation of cadaveric pancreatic organs or pancreatic islets can restore normal physiology. However, there is a chronic shortage of cadaveric organs, limiting the treatment of the majority of patients on the pancreas transplantation waiting list. Here, we hypothesized that human iPS cells can be directly differentiated into insulin producing cells (IPCs) capable of secreting insulin. Using a series of pancreatic growth factors, we successfully generated iPS cells derived IPCs. Furthermore, to investigate the capability of these cells to secrete insulin in vivo, the differentiated cells were transplanted under the kidney capsules of diabetic immunodeficient mice. Serum glucose levels gradually declined to either normal or near normal levels over 150 days, suggesting that the IPCs were secreting insulin. In addition, using MRI, a 3D organoid appeared as a white patch on the transplanted kidneys but not on the control kidneys. These organoids showed neo-vascularization and stained positive for insulin and glucagon. All together, these data show that a pancreatic organ can be created in vivo providing evidence that iPS cells might be a novel option for the treatment of T1D.
Assuntos
Diferenciação Celular , Células-Tronco Pluripotentes Induzidas/citologia , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/metabolismo , Neovascularização Fisiológica , Transplante de Células-Tronco , Animais , Glicemia , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/terapia , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/ultraestrutura , Imageamento por Ressonância Magnética , Masculino , Camundongos , Camundongos Knockout , Mitocôndrias/metabolismo , Organoides , Consumo de OxigênioRESUMO
Gene therapy using non-viral vectors that are safe and efficient in transfecting target cells is an effective approach to overcome the shortcomings of protein delivery of growth factors. The objective of this study was to develop and test a non-viral gene delivery system for bone regeneration utilizing a collagen scaffold to deliver polyethylenimine (PEI)-plasmid DNA (pDNA) [encoding platelet derived growth factor-B (PDGF-B)] complexes. The PEI-pPDGF-B complexes were fabricated at amine (N) to phosphate (P) ratio of 10 and characterized for size, surface charge, and in vitro cytotoxicity and transfection efficacy in human bone marrow stromal cells (BMSCs). The influence of the complex-loaded collagen scaffold on cellular attachment and recruitment was evaluated in vitro using microscopy techniques. The in vivo regenerative capacity of the gene delivery system was assessed in 5 mm diameter critical-sized calvarial defects in Fisher 344 rats. The complexes were ~100 nm in size with a positive surface charge. Complexes prepared at an N/P ratio of 10 displayed low cytotoxicity as assessed by a cell viability assay. Confocal microscopy revealed significant proliferation of BMSCs on complex-loaded collagen scaffolds compared to empty scaffolds. In vivo studies showed significantly higher new bone volume/total volume (BV/TV) % in calvarial defects treated with the complex-activated scaffolds following 4 weeks of implantation (14- and 44-fold higher) when compared to empty defects or empty scaffolds, respectively. Together, these findings suggest that non-viral PDGF-B gene-activated scaffolds are effective for bone regeneration and are an attractive gene delivery system with significant potential for clinical translation.
Assuntos
Regeneração Óssea/genética , Técnicas de Transferência de Genes , Fator de Crescimento Derivado de Plaquetas/genética , Alicerces Teciduais/química , Animais , Proliferação de Células , Sobrevivência Celular , Colágeno/química , DNA , Expressão Gênica , Terapia Genética , Vetores Genéticos , Humanos , Masculino , Células-Tronco Mesenquimais , Microscopia Confocal , Plasmídeos/genética , Fator de Crescimento Derivado de Plaquetas/metabolismo , Polietilenoimina , Ratos , Ratos Endogâmicos F344 , TransfecçãoRESUMO
Increased reactive oxygen species (ROS) contribute to asthma, but little is known about the molecular mechanisms connecting increased ROS with characteristic features of asthma. We show that enhanced oxidative activation of the Ca(2+)/calmodulin-dependent protein kinase (ox-CaMKII) in bronchial epithelium positively correlates with asthma severity and that epithelial ox-CaMKII increases in response to inhaled allergens in patients. We used mouse models of allergic airway disease induced by ovalbumin (OVA) or Aspergillus fumigatus (Asp) and found that bronchial epithelial ox-CaMKII was required to increase a ROS- and picrotoxin-sensitive Cl(-) current (ICl) and MUC5AC expression, upstream events in asthma progression. Allergen challenge increased epithelial ROS by activating NADPH oxidases. Mice lacking functional NADPH oxidases due to knockout of p47 and mice with epithelial-targeted transgenic expression of a CaMKII inhibitory peptide or wild-type mice treated with inhaled KN-93, an experimental small-molecule CaMKII antagonist, were protected against increases in ICl, MUC5AC expression, and airway hyperreactivity to inhaled methacholine. Our findings support the view that CaMKII is a ROS-responsive, pluripotent proasthmatic signal and provide proof-of-concept evidence that CaMKII is a therapeutic target in asthma.
Assuntos
Asma/enzimologia , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Administração Intranasal , Animais , Asma/tratamento farmacológico , Asma/metabolismo , Benzilaminas/administração & dosagem , Benzilaminas/uso terapêutico , Western Blotting , Brônquios/metabolismo , Brônquios/patologia , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/antagonistas & inibidores , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Feminino , Humanos , Técnicas In Vitro , Masculino , Camundongos , NADPH Oxidases/metabolismo , Ovalbumina/farmacologia , Oxirredução , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/uso terapêutico , Espécies Reativas de Oxigênio/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Sulfonamidas/administração & dosagem , Sulfonamidas/uso terapêuticoRESUMO
Myocardial cell death is initiated by excessive mitochondrial Ca(2+) entry causing Ca(2+) overload, mitochondrial permeability transition pore (mPTP) opening and dissipation of the mitochondrial inner membrane potential (ΔΨm). However, the signalling pathways that control mitochondrial Ca(2+) entry through the inner membrane mitochondrial Ca(2+) uniporter (MCU) are not known. The multifunctional Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) is activated in ischaemia reperfusion, myocardial infarction and neurohumoral injury, common causes of myocardial death and heart failure; these findings suggest that CaMKII could couple disease stress to mitochondrial injury. Here we show that CaMKII promotes mPTP opening and myocardial death by increasing MCU current (I(MCU)). Mitochondrial-targeted CaMKII inhibitory protein or cyclosporin A, an mPTP antagonist with clinical efficacy in ischaemia reperfusion injury, equivalently prevent mPTP opening, ΔΨm deterioration and diminish mitochondrial disruption and programmed cell death in response to ischaemia reperfusion injury. Mice with myocardial and mitochondrial-targeted CaMKII inhibition have reduced I(MCU) and are resistant to ischaemia reperfusion injury, myocardial infarction and neurohumoral injury, suggesting that pathological actions of CaMKII are substantially mediated by increasing I(MCU). Our findings identify CaMKII activity as a central mechanism for mitochondrial Ca(2+) entry in myocardial cell death, and indicate that mitochondrial-targeted CaMKII inhibition could prevent or reduce myocardial death and heart failure in response to common experimental forms of pathophysiological stress.
Assuntos
Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Cálcio/metabolismo , Mitocôndrias Cardíacas/metabolismo , Mitocôndrias Cardíacas/patologia , Miocárdio/enzimologia , Miocárdio/patologia , Estresse Fisiológico , Animais , Apoptose/efeitos dos fármacos , Cálcio/farmacologia , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/antagonistas & inibidores , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/química , Ciclosporina/farmacologia , Feminino , Coração/efeitos dos fármacos , Coração/fisiopatologia , Insuficiência Cardíaca/tratamento farmacológico , Insuficiência Cardíaca/prevenção & controle , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Potencial da Membrana Mitocondrial/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mitocôndrias Cardíacas/enzimologia , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Poro de Transição de Permeabilidade Mitocondrial , Infarto do Miocárdio/tratamento farmacológico , Infarto do Miocárdio/prevenção & controle , Miocárdio/metabolismo , Traumatismo por Reperfusão/enzimologia , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/patologia , Traumatismo por Reperfusão/prevenção & controle , Serina/metabolismo , Estresse Fisiológico/efeitos dos fármacosRESUMO
BACKGROUND: A previous study showed that running polypropylene sutures anchored with square knots retain only 75% of their strength compared with half hitches. The aim of this study was to investigate whether anchor knot geometry similarly affects the tensile strength of other types of sutures used in continuous closures. METHODS: Monofilament and multifilament sutures (all 3-0) were anchored with either square knots or half hitches to 1 tensionometer post, and the running ends were secured to the other. The force required to break the running suture and the site of suture failure were recorded. RESULTS: The running sutures anchored with square knots retained only 50% to 84% of the strength of the identical sutures secured with half hitches (P < .001). CONCLUSIONS: A running suture anchored with half hitches is stronger and safer in comparison with the same suture anchored with square knots. This study provokes a fundamental reconsideration of the use of square knots to anchor running sutures.
Assuntos
Técnicas de Sutura , Suturas , Categute , Dioxanos , Humanos , Microscopia Eletrônica de Varredura , Nylons , Polidioxanona , Poliésteres , Poliglactina 910 , Seda , Resistência à TraçãoRESUMO
Excessive activation of the ß-adrenergic, angiotensin II (Ang II) and aldosterone signaling pathways promotes mortality after myocardial infarction, and antagonists targeting these pathways are core therapies for treating this condition. Catecholamines and Ang II activate the multifunctional Ca(2+)/calmodulin-dependent protein kinase II (CaMKII), the inhibition of which prevents isoproterenol-mediated and Ang II-mediated cardiomyopathy. Here we show that aldosterone exerts direct toxic actions on myocardium by oxidative activation of CaMKII, causing cardiac rupture and increased mortality in mice after myocardial infarction. Aldosterone induces CaMKII oxidation by recruiting NADPH oxidase, and this oxidized and activated CaMKII promotes matrix metalloproteinase 9 (MMP9) expression in cardiomyocytes. Myocardial CaMKII inhibition, overexpression of methionine sulfoxide reductase A (an enzyme that reduces oxidized CaMKII) or NADPH oxidase deficiency prevented aldosterone-enhanced cardiac rupture after myocardial infarction. These findings show that oxidized myocardial CaMKII mediates the cardiotoxic effects of aldosterone on the cardiac matrix and establish CaMKII as a nodal signal for the neurohumoral pathways associated with poor outcomes after myocardial infarction.