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1.
Arch Toxicol ; 97(7): 1847-1858, 2023 07.
Artigo em Inglês | MEDLINE | ID: mdl-37166470

RESUMO

Arsenic trioxide (ATO), an inorganic arsenical, is a toxic environmental contaminant. It is also a widely used chemical with industrial and medicinal uses. Significant public health risk exists from its intentional or accidental exposure. The pulmonary pathology of acute high dose exposure is not well defined. We developed and characterized a murine model of a single inhaled exposure to ATO, which was evaluated 24 h post-exposure. ATO caused hypoxemia as demonstrated by arterial blood-gas measurements. ATO administration caused disruption of alveolar-capillary membrane as shown by increase in total protein and IgM in the bronchoalveolar lavage fluid (BALF) supernatant and an onset of pulmonary edema. BALF of ATO-exposed mice had increased HMGB1, a damage-associated molecular pattern (DAMP) molecule, and differential cell counts revealed increased neutrophils. BALF supernatant also showed an increase in protein levels of eotaxin/CCL-11 and MCP-3/CCL-7 and a reduction in IL-10, IL-19, IFN-γ, and IL-2. In the lung of ATO-exposed mice, increased protein levels of G-CSF, CXCL-5, and CCL-11 were noted. Increased mRNA levels of TNF-a, and CCL2 in ATO-challenged lungs further supported an inflammatory pathogenesis. Neutrophils were increased in the blood of ATO-exposed animals. Pulmonary function was also evaluated using flexiVent. Consistent with an acute lung injury phenotype, respiratory and lung elastance showed significant increase in ATO-exposed mice. PV loops showed a downward shift and a decrease in inspiratory capacity in the ATO mice. Flow-volume curves showed a decrease in FEV0.1 and FEF50. These results demonstrate that inhaled ATO leads to pulmonary damage and characteristic dysfunctions resembling ARDS in humans.


Assuntos
Lesão Pulmonar Aguda , Arsenicais , Humanos , Camundongos , Animais , Modelos Animais de Doenças , Pulmão/patologia , Líquido da Lavagem Broncoalveolar/química
2.
Arch Toxicol ; 95(1): 179-193, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-32979061

RESUMO

Accidental bromine spills are common and its large industrial stores risk potential terrorist attacks. The mechanisms of bromine toxicity and effective therapeutic strategies are unknown. Our studies demonstrate that inhaled bromine causes deleterious cardiac manifestations. In this manuscript we describe mechanisms of delayed cardiac effects in the survivors of a single bromine exposure. Rats were exposed to bromine (600 ppm for 45 min) and the survivors were sacrificed at 14 or 28 days. Echocardiography, hemodynamic analysis, histology, transmission electron microscopy (TEM) and biochemical analysis of cardiac tissue were performed to assess functional, structural and molecular effects. Increases in right ventricular (RV) and left ventricular (LV) end-diastolic pressure and LV end-diastolic wall stress with increased LV fibrosis were observed. TEM images demonstrated myofibrillar loss, cytoskeletal breakdown and mitochondrial damage at both time points. Increases in cardiac troponin I (cTnI) and N-terminal pro brain natriuretic peptide (NT-proBNP) reflected myofibrillar damage and increased LV wall stress. LV shortening decreased as a function of increasing LV end-systolic wall stress and was accompanied by increased sarcoendoplasmic reticulum calcium ATPase (SERCA) inactivation and a striking dephosphorylation of phospholamban. NADPH oxidase 2 and protein phosphatase 1 were also increased. Increased circulating eosinophils and myocardial 4-hydroxynonenal content suggested increased oxidative stress as a key contributing factor to these effects. Thus, a continuous oxidative stress-induced chronic myocardial damage along with phospholamban dephosphorylation are critical for bromine-induced chronic cardiac dysfunction. These findings in our preclinical model will educate clinicians and public health personnel and provide important endpoints to evaluate therapies.


Assuntos
Bromo , Cardiomegalia/fisiopatologia , Disfunção Ventricular Esquerda/fisiopatologia , Disfunção Ventricular Direita/fisiopatologia , Função Ventricular Esquerda , Função Ventricular Direita , Remodelação Ventricular , Animais , Proteínas de Ligação ao Cálcio/metabolismo , Cardiomegalia/induzido quimicamente , Cardiomegalia/metabolismo , Cardiomegalia/patologia , Cardiotoxicidade , Diástole , Modelos Animais de Doenças , Fibrose , Masculino , Mitocôndrias Cardíacas/metabolismo , Mitocôndrias Cardíacas/ultraestrutura , Miocárdio/metabolismo , Miocárdio/ultraestrutura , NADPH Oxidase 2/metabolismo , Peptídeo Natriurético Encefálico/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Fragmentos de Peptídeos/metabolismo , Fosforilação , Proteína Fosfatase 1/metabolismo , Ratos Sprague-Dawley , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Sístole , Fatores de Tempo , Troponina I/metabolismo , Disfunção Ventricular Esquerda/induzido quimicamente , Disfunção Ventricular Esquerda/metabolismo , Disfunção Ventricular Esquerda/patologia , Disfunção Ventricular Direita/induzido quimicamente , Disfunção Ventricular Direita/metabolismo , Disfunção Ventricular Direita/patologia
3.
Ann N Y Acad Sci ; 1479(1): 210-222, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32329907

RESUMO

Lewisite is a strong vesicating and chemical warfare agent. Because of the rapid transdermal absorption, cutaneous exposure to lewisite can also elicit severe systemic injury. Lewisite (2.5, 5.0, and 7.5 mg/kg) was applied to the skin of Ptch1+/- /SKH-1 mice and acute lung injury (ALI) was assessed after 24 hours. Arterial blood gas measurements showed hypercapnia and hypoxemia in the lewisite-exposed group. Histological evaluation of lung tissue revealed increased levels of proinflammatory neutrophils and a dose-dependent increase in structural changes indicative of injury. Increased inflammation was also confirmed by altered expression of cytokines, including increased IL-33, and a dose-dependent elevation of CXCL1, CXCL5, and GCSF was observed in the lung tissue. In the bronchoalveolar lavage fluid of lewisite-exposed animals, there was a significant increase in HMGB1, a damage-associated molecular pattern molecule, as well as elevated CXCL1 and CXCL5, which coincided with an influx of neutrophils to the lungs. Complete blood cell analysis revealed eosinophilia and altered neutrophil-lymphocyte ratios as a consequence of lewisite exposure. Mean platelet volume and RBC distribution width, which are predictors of lung injury, were also increased in the lewisite group. These data demonstrate that cutaneous lewisite exposure causes ALI and may contribute to mortality in exposed populations.


Assuntos
Lesão Pulmonar Aguda , Arsenicais , Substâncias para a Guerra Química/intoxicação , Citocinas/metabolismo , Pulmão , Infiltração de Neutrófilos/efeitos dos fármacos , Neutrófilos , Lesão Pulmonar Aguda/induzido quimicamente , Lesão Pulmonar Aguda/metabolismo , Lesão Pulmonar Aguda/patologia , Animais , Lavagem Broncoalveolar , Feminino , Contagem de Leucócitos , Pulmão/metabolismo , Pulmão/patologia , Masculino , Camundongos , Camundongos Pelados , Neutrófilos/metabolismo , Neutrófilos/patologia , Contagem de Plaquetas , Pele/metabolismo , Pele/patologia
4.
Heliyon ; 5(4): e01397, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30997426

RESUMO

BACKGROUND: Volume overload (VO) of isolated mitral regurgitation (MR) or aortocaval fistula (ACF) is associated with extracellular matrix degradation and cardiomyocyte myofibrillar and desmin breakdown. Left ventricular (LV) chymase activity is increased in VO and recent studies demonstrate chymase presence within cardiomyocytes. Here we test the hypothesis that chymase within the cardiomyocyte coincides with myosin and desmin breakdown in VO. METHODS AND RESULTS: Aortocaval fistula (ACF) was induced in Sprague Dawley (SD) rats and was compared to age-matched sham-operated rats at 24 hours, 4 and 12 weeks. Immunohistochemistry (IHC) and transmission electron microscopy (TEM) immunogold of LV tissue demonstrate chymase within cardiomyocytes at all ACF time points. IHC for myosin demonstrates myofibrillar disorganization starting at 24 hours. Proteolytic presence of chymase in cardiomyocytes is verified by in situ chymotryptic tissue activity that is inhibited by pretreatment with a chymase inhibitor. Real-time PCR of isolated cardiomyocytes at all ACF time points and in situ hybridization demonstrate endothelial cells and fibroblasts as a major source of chymase mRNA in addition to mast cells. Chymase added to adult rat cardiomyocytes in vitro is taken up by a dynamin-mediated process and myosin breakdown is attenuated by dynamin inhibitor, suggesting that chymase uptake is essential for myosin breakdown. In a previous study in the dog model of chronic MR, the intracellular changes were attributed to extracellular effects. However, we now demonstrate intracellular effects of chymase in both species. CONCLUSION: In response to VO, fibroblast and endothelial cells produce chymase and subsequent cardiomyocyte chymase uptake is followed by myosin degradation. The results demonstrate a novel intracellular chymase-mediated mechanism of cardiomyocyte dysfunction and adverse remodeling in a pure VO.

5.
Am J Physiol Heart Circ Physiol ; 316(1): H212-H223, 2019 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-30379573

RESUMO

Halogens are widely used, highly toxic chemicals that pose a potential threat to humans because of their abundance. Halogens such as bromine (Br2) cause severe pulmonary and systemic injuries; however, the mechanisms of their toxicity are largely unknown. Here, we demonstrated that Br2 and reactive brominated species produced in the lung and released in blood reach the heart and cause acute cardiac ultrastructural damage and dysfunction in rats. Br2-induced cardiac damage was demonstrated by acute (3-24 h) increases in circulating troponin I, heart-type fatty acid-binding protein, and NH2-terminal pro-brain natriuretic peptide. Transmission electron microscopy demonstrated acute (3-24 h) cardiac contraction band necrosis, disruption of z-disks, and mitochondrial swelling and disorganization. Echocardiography and hemodynamic analysis revealed left ventricular (LV) systolic and diastolic dysfunction at 7 days. Plasma and LV tissue had increased levels of brominated fatty acids. 2-Bromohexadecanal (Br-HDA) injected into the LV cavity of a normal rat caused acute LV enlargement with extensive disruption of the sarcomeric architecture and mitochondrial damage. There was extensive infiltration of neutrophils and increased myeloperoxidase levels in the hearts of Br2- or Br2 reactant-exposed rats. Increased bromination of sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) and increased phosphalamban after Br2 inhalation decreased cardiac SERCA activity by 70%. SERCA inactivation was accompanied by increased Ca2+-sensitive LV calpain activity. The calpain-specific inhibitor MDL28170 administered within 1 h after exposure significantly decreased calpain activity and acute mortality. Bromine inhalation and formation of reactive brominated species caused acute cardiac injury and myocardial damage that can lead to heart failure. NEW & NOTEWORTHY The present study defines left ventricular systolic and diastolic dysfunction due to cardiac injury after bromine (Br2) inhalation. A calpain-dependent mechanism was identified as a potential mediator of cardiac ultrastructure damage. This study not only highlights the importance of monitoring acute cardiac symptoms in victims of Br2 exposure but also defines calpains as a potential target to treat Br2-induced toxicity.


Assuntos
Bromo/toxicidade , Calpaína/metabolismo , Traumatismo por Reperfusão Miocárdica/etiologia , Miócitos Cardíacos/efeitos dos fármacos , Disfunção Ventricular/etiologia , Administração por Inalação , Animais , Biomarcadores/sangue , Bromo/administração & dosagem , Células Cultivadas , Hemodinâmica , Masculino , Mitocôndrias Cardíacas/efeitos dos fármacos , Mitocôndrias Cardíacas/metabolismo , Mitocôndrias Cardíacas/patologia , Contração Miocárdica , Traumatismo por Reperfusão Miocárdica/metabolismo , Traumatismo por Reperfusão Miocárdica/patologia , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Ratos , Ratos Sprague-Dawley , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Disfunção Ventricular/metabolismo , Disfunção Ventricular/patologia , Remodelação Ventricular
6.
Am J Physiol Lung Cell Mol Physiol ; 316(1): L94-L104, 2019 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-30358437

RESUMO

Nicotine is a highly addictive principal component of both tobacco and electronic cigarette that is readily absorbed in blood. Nicotine-containing electronic cigarettes are promoted as a safe alternative to cigarette smoking. However, the isolated effects of inhaled nicotine are largely unknown. Here we report a novel rat model of aerosolized nicotine with a particle size (~1 µm) in the respirable diameter range. Acute nicotine inhalation caused increased pulmonary edema and lung injury as measured by enhanced bronchoalveolar lavage fluid protein, IgM, lung wet-to-dry weight ratio, and high-mobility group box 1 (HMGB1) protein and decreased lung E-cadherin protein. Immunohistochemical analysis revealed congested blood vessels and increased neutrophil infiltration. Lung myeloperoxidase mRNA and protein increased in the nicotine-exposed rats. Complete blood counts also showed an increase in neutrophils, white blood cells, eosinophils, and basophils. Arterial blood gas measurements showed an increase in lactate. Lungs of nicotine-inhaling animals revealed increased mRNA levels of IL-1A and CXCL1. There was also an increase in IL-1α protein. In in vitro air-liquid interface cultures of airway epithelial cells, there was a dose dependent increase in HMGB1 release with nicotine treatment. Air-liquid cultures exposed to nicotine also resulted in a dose-dependent loss of barrier as measured by transepithelial electrical resistance and a decrease in E-cadherin expression. Nicotine also caused a dose-dependent increase in epithelial cell death and an increase in caspase-3/7 activities. These results show that the nicotine content of electronic cigarettes may have adverse pulmonary and systemic effects.


Assuntos
Barreira Alveolocapilar , Nicotina/efeitos adversos , Vaping , Aerossóis , Animais , Barreira Alveolocapilar/lesões , Barreira Alveolocapilar/metabolismo , Barreira Alveolocapilar/patologia , Caspase 3/metabolismo , Caspase 7/metabolismo , Quimiocina CXCL1/sangue , Proteína HMGB1/metabolismo , Imunoglobulina M/sangue , Interleucina-1alfa/sangue , Contagem de Leucócitos , Masculino , Infiltração de Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismo , Neutrófilos/patologia , Nicotina/farmacologia , Tamanho da Partícula , Edema Pulmonar/sangue , Edema Pulmonar/induzido quimicamente , Edema Pulmonar/patologia , Ratos , Ratos Sprague-Dawley , Vaping/efeitos adversos , Vaping/sangue , Vaping/patologia
7.
Oncol Lett ; 15(4): 5105-5110, 2018 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-29552145

RESUMO

Toll-like receptor (TLR) 7/8 agonists have been applied in combination with chemo-, radio- or immunotherapy for lymphoma, and used as topical drugs for the treatment of viral skin lesions and skin tumors. In the present study, the role of an adenine analog, 9-(4-carboxyphenyl)-8-hydroxy-2-(2-methoxyethoxy)-adenine [termed Gao Dong (GD)], a novel TLR7 agonist, in the activation of cytokine-induced killer/natural killer (CIK/NK) cells was determined. The results of the present study indicated that GD was able to activate CIK/NK cells. The proportion of GD-induced CD3+CD56+ CIK and CD3-CD56+ NK cells was ~4% higher respectively compared with the control. Notably, combination therapy with CIK/NK cells stimulated by GD, markedly suppressed the proliferation of the chronic myelogenous leukemia K562 cell line. Following GD treatment, the cytotoxicity improved by ~25 and 21% when the effector/target ratio was 20:1 and 10:1, respectively. The results of the present study suggested a novel protocol for CIK/NK cell proliferation and revealed that GD may serve as a potent innate and adaptive immunomodulator in immunocyte culture.

8.
Curr Eye Res ; 43(6): 810-820, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29505314

RESUMO

PURPOSE: The purpose of this study is to investigate the potential therapeutic benefits of intravitreally transplanted human umbilical cord mesenchymal stem cells (UC-MSCs) in an animal model of microbead-injection-induced ocular hypertension (OHT). METHODS: UC-MSCs were isolated from human umbilical cords and then cultured. The OHT model was induced via intracameral injection of polystyrene microbeads in Sprague-Dawley adult rat eyes. Fifty-four healthy adult rats were randomly divided into three groups: normal control, OHT model treated with intravitreal transplantation of UC-MSCs, or phosphate-buffered saline (PBS). Two days after OHT was induced, either 5 µl 105 UC-MSCs suspension or PBS was injected into the vitreous cavity of rats. UC-MSCs localization and integration were examined via immunohistochemistry. Neuroprotection was quantified by counting retinal ganglion cells (RGCs) and axons 2 weeks following transplantation. The expression levels of glial-derived neurotrophic factor (GDNF), brain-derived neurotrophic factor (BDNF), and glial fibrillary acidic protein (GFAP) were assessed via immunohistochemistry and Western blot. Functional recovery was assessed 2 weeks after transplantation via scotopic threshold response (STR) electroretinography. RESULTS: Elevated IOP levels were sustained at least 3 weeks after intracameral microbead injection and the number of ß-III-tubulin+ RGCs significantly declined compared to PBS-injected eyes. UC-MSCs survived for at least 2 weeks after intravitreal transplantation and predominantly located in the vitreous cavity. A fraction of cells migrated into the ganglion cell layer of host retina, but without differentiation. Intravitreal UC-MSC transplantation resulted in increased number of RGCs, axons, and increased expression of GDNF and BDNF but decreased expression of GFAP. Intravitreal delivery of UC-MSCs significantly improved the recovery of the positive STR. CONCLUSIONS: Intravitreal transplantation of UC-MSCs revealed the neuroprotection in the microbead-injection induced OHT. The effects could be related to the secretion of tropic factors (BDNF and GDNF) and the modulation of glial cell activation.


Assuntos
Pressão Intraocular/fisiologia , Transplante de Células-Tronco Mesenquimais/métodos , Células-Tronco Mesenquimais/citologia , Neuroproteção , Hipertensão Ocular/terapia , Cordão Umbilical/citologia , Animais , Contagem de Células , Diferenciação Celular , Modelos Animais de Doenças , Masculino , Microesferas , Hipertensão Ocular/diagnóstico , Hipertensão Ocular/fisiopatologia , Ratos , Ratos Sprague-Dawley , Células Ganglionares da Retina/patologia
10.
J Cancer ; 8(16): 3183-3189, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29158790

RESUMO

Chemo- and radio-therapy suffer from certain well-recognized drawbacks for lymphoma therapy. Passive immunotherapy with monoclonal antibody has improved outcome for patients with CD20+ B cell lymphoma, but not for T cell lymphoma. Therefore, novel treatment approaches are clearly required for T cell lymphoma. To date, the combined application targeting TLR7, 8 and 9 has established long-term antitumor immunity. We previously synthesized a purine-scaffold TLR7 agonist named GD5. Here, we report that the intratumoral administration of GD5 combined with doxorubicin (DOX), a conventional chemotherapeutic agent in T cell lymphoma. This combined treatment made mice to produce more cytokines in blood, and generate more potent cytotoxic T lymphocyte response, then result in effective eradication of both local and distant tumors in tumor-bearing mice. Our findings demonstrate the potential for enhancing the efficacy of the current standard DOX therapy through combination with TLR7 agonist GD5 to improve antitumor immune responses and provide durable remissions for T cell lymphoma.

11.
Am J Physiol Heart Circ Physiol ; 313(1): H32-H45, 2017 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-28455287

RESUMO

Heart failure due to chronic volume overload (VO) in rats and humans is characterized by disorganization of the cardiomyocyte desmin/mitochondrial network. Here, we tested the hypothesis that desmin breakdown is an early and continuous process throughout VO. Male Sprague-Dawley rats had aortocaval fistula (ACF) or sham surgery and were examined 24 h and 4 and 12 wk later. Desmin/mitochondrial ultrastructure was examined by transmission electron microscopy (TEM) and immunohistochemistry (IHC). Protein and kinome analysis were performed in isolated cardiomyocytes, and desmin cleavage was assessed by mass spectrometry in left ventricular (LV) tissue. Echocardiography demonstrated a 40% decrease in the LV mass-to-volume ratio with spherical remodeling at 4 wk with ACF and LV systolic dysfunction at 12 wk. Starting at 24 h and continuing to 4 and 12 wk, with ACF there is TEM evidence of extensive mitochondrial clustering, IHC evidence of disorganization associated with desmin breakdown, and desmin protein cleavage verified by Western blot analysis and mass spectrometry. IHC results revealed that ACF cardiomyocytes at 4 and 12 wk had perinuclear translocation of αB-crystallin from the Z disk with increased α, ß-unsaturated aldehyde 4-hydroxynonelal. Use of protein markers with verification by TUNEL staining and kinome analysis revealed an absence of cardiomyocyte apoptosis at 4 and 12 wk of ACF. Significant increases in protein indicators of mitophagy were countered by a sixfold increase in p62/sequestosome-1, which is indicative of an inability to complete autophagy. An early and continuous disruption of the desmin/mitochondrial architecture, accompanied by oxidative stress and inhibition of apoptosis and mitophagy, suggests its causal role in LV dilatation and systolic dysfunction in VO.NEW & NOTEWORTHY This study provides new evidence of early onset (24 h) and continuous (4-12 wk) desmin misarrangement and disruption of the normal sarcomeric and mitochondrial architecture throughout the progression of volume overload heart failure, suggesting a causal link between desmin cleavage and mitochondrial disorganization and damage.


Assuntos
Desmina/metabolismo , Insuficiência Cardíaca/patologia , Insuficiência Cardíaca/fisiopatologia , Mitocôndrias Cardíacas/ultraestrutura , Disfunção Ventricular Esquerda/patologia , Disfunção Ventricular Esquerda/fisiopatologia , Animais , Apoptose , Células Cultivadas , Doença Crônica , Insuficiência Cardíaca/complicações , Masculino , Mitocôndrias Cardíacas/metabolismo , Mitocôndrias Cardíacas/patologia , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Miócitos Cardíacos/ultraestrutura , Estresse Oxidativo , Ratos , Ratos Sprague-Dawley , Disfunção Ventricular Esquerda/complicações
12.
J Mol Cell Cardiol ; 92: 1-9, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26807691

RESUMO

BACKGROUND: Previous work has identified mast cells as the major source of chymase largely associated with a profibrotic phenotype. We recently reported increased fibroblast autophagic procollagen degradation in a rat model of pure volume overload (VO). Here we demonstrate a connection between increased fibroblast chymase production and autophagic digestion of procollagen in the pure VO of aortocaval fistula (ACF) in the rat. METHODS AND RESULTS: Isolated LV fibroblasts taken from 4 and 12week ACF Sprague-Dawley rats have significant increases in chymase mRNA and chymase activity. Increased intracellular chymase protein is documented by immunocytochemistry in the ACF fibroblasts compared to cells obtained from age-matched sham rats. To implicate VO as a stimulus for chymase production, we show that isolated adult rat LV fibroblasts subjected to 24h of 20% cyclical stretch induces chymase mRNA and protein production. Exogenous chymase treatment of control isolated adult cardiac fibroblasts demonstrates that chymase is internalized through a dynamin-dependent mechanism. Chymase treatment leads to an increased formation of autophagic vacuoles, LC3-II production, autophagic flux, resulting in increased procollagen degradation. Chymase inhibitor treatment reduces cyclical stretch-induced autophagy in isolated cardiac fibroblasts, demonstrating chymase's role in autophagy induction. CONCLUSION: In a pure VO model, chymase produced in adult cardiac fibroblasts leads to autophagic degradation of newly synthesized intracellular procollagen I, suggesting a new role of chymase in extracellular matrix degradation.


Assuntos
Aorta/metabolismo , Quimases/biossíntese , Insuficiência Cardíaca/metabolismo , Pró-Colágeno/metabolismo , Animais , Aorta/patologia , Fístula Artério-Arterial , Autofagia/genética , Quimases/metabolismo , Matriz Extracelular/metabolismo , Fibroblastos/metabolismo , Fibroblastos/patologia , Insuficiência Cardíaca/patologia , Humanos , Mastócitos/metabolismo , Mastócitos/patologia , Miocárdio/metabolismo , Miocárdio/patologia , Fagossomos/metabolismo , Proteólise , RNA Mensageiro/biossíntese , Ratos
13.
J Mol Cell Cardiol ; 89(Pt B): 241-250, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26596413

RESUMO

In a pure volume overloaded (VO) heart, interstitial collagen loss is degraded by matrix metalloproteinases (MMPs) that leads to left ventricular (LV) dilatation and heart failure. Cardiac fibroblasts are the primary source of extracellular matrix proteins that connect cardiomyocytes. The goal of this study was to determine how VO affects intracellular procollagen in cardiac fibroblasts. Using the aortocaval fistula (ACF) model in Sprague-Dawley rats, we demonstrate that cardiac fibroblasts isolated from 4 and 12 wk ACF animals have decreased intracellular procollagen I compared to the fibroblasts from age-matched shams. The reduction of procollagen I is associated with increased autophagy as demonstrated by increased autophagic vacuoles and LC3-II expression. To test the relationship between autophagy and procollagen degradation, we treated adult cardiac fibroblasts with either an autophagy inducer, rapamycin, or an inhibitor, wortmannin, and found that procollagen I protein levels were decreased in fibroblasts treated with rapamycin and elevated in wortmannin-treated cells. In addition, we demonstrated that VO induces oxidative stresses in cardiac fibroblasts from 4 and 12 wk ACF rats. Treatment of cultured cardiac fibroblasts with an oxidative stress-inducing agent (DMNQ) induces autophagy and intracellular procollagen I and fibronectin degradation, which is reversed by wortmannin but not by the global MMP inhibitor (PD166793). Mechanical stretch of cardiac fibroblasts also induces oxidative stress and autophagic degradation of procollagen I and fibronectin. Our results suggest that in addition to the well-known effects of MMPs on extracellular collagen degradation in VO, there is a concurrent degradation of intracellular procollagen and fibronectin mediated by oxidative stress-induced autophagy in cardiac fibroblasts.


Assuntos
Autofagia , Colágeno/metabolismo , Fibroblastos/metabolismo , Fibroblastos/patologia , Miocárdio/patologia , Proteólise , Animais , Peso Corporal , Separação Celular , Ativação Enzimática , Fibroblastos/ultraestrutura , Fibronectinas/metabolismo , Frequência Cardíaca , Masculino , Metaloproteinase 2 da Matriz/metabolismo , Estresse Oxidativo , Ratos Sprague-Dawley , Estresse Mecânico , Vacúolos/metabolismo , Vacúolos/ultraestrutura , Fístula Vascular/patologia , Fístula Vascular/fisiopatologia , Função Ventricular Esquerda , Remodelação Ventricular
14.
Cell Transplant ; 23(4-5): 505-12, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24816447

RESUMO

Human bone marrow-derived mesenchymal stem cells (MSCs) can self-renew and differentiate into osteoblasts, chondrocytes, and adipocytes. MSCs have effectively emerged as a promising tool for clinical applications, specifically in musculoskeletal diseases. This article reviews the status of preclinical animal studies, clinical trials, and the efforts of the industry in using MSCs to treat musculoskeletal diseases such as bone fractures, bone defects, focal chondral lesions, osteoarthritis, spinal diseases, and tendon injuries. We also discuss the current problems encountered and potential of using MSCs in future clinical studies.


Assuntos
Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/citologia , Doenças Musculoesqueléticas/terapia , Animais , Células da Medula Óssea/citologia , Regeneração Óssea , Diferenciação Celular , Indústria Farmacêutica , Humanos , Medicina Regenerativa , Regeneração da Medula Espinal
15.
PLoS One ; 9(4): e94732, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24733352

RESUMO

Cardiac ischemia and reperfusion (I/R) injury occurs because the acute increase in oxidative/inflammatory stress during reperfusion culminates in the death of cardiomyocytes. Currently, there is no drug utilized clinically that attenuates I/R injury in patients. Previous studies have demonstrated degranulation of mast cell contents into the interstitium after I/R. Using a dog model of I/R, we tested the role of chymase, a mast cell protease, in cardiomyocyte injury using a specific oral chymase inhibitor (CI). 15 adult mongrel dogs had left anterior descending artery occlusion for 60 min and reperfusion for 100 minutes. 9 dogs received vehicle and 6 were pretreated with a specific CI. In vivo cardiac microdialysis demonstrated a 3-fold increase in interstitial fluid chymase activity in I/R region that was significantly decreased by CI. CI pretreatment significantly attenuated loss of laminin, focal adhesion complex disruption, and release of troponin I into the circulation. Microarray analysis identified an I/R induced 17-fold increase in nuclear receptor subfamily 4A1 (NR4A1) and significantly decreased by CI. NR4A1 normally resides in the nucleus but can induce cell death on migration to the cytoplasm. I/R caused significant increase in NR4A1 protein expression and cytoplasmic translocation, and mitochondrial degradation, which were decreased by CI. Immunohistochemistry also revealed a high concentration of chymase within cardiomyocytes after I/R. In vitro, chymase added to culture HL-1 cardiomyocytes entered the cytoplasm and nucleus in a dynamin-dependent fashion, and promoted cytoplasmic translocation of NR4A1 protein. shRNA knockdown of NR4A1 on pre-treatment of HL-1 cells with CI significantly decreased chymase-induced cell death and mitochondrial damage. These results suggest that the beneficial effects of an orally active CI during I/R are mediated in the cardiac interstitium as well as within the cardiomyocyte due to a heretofore-unrecognized chymase entry into cardiomyocytes.


Assuntos
Quimases/fisiologia , Mitocôndrias/metabolismo , Miócitos Cardíacos/enzimologia , Ferimentos e Lesões/metabolismo , Animais , Linhagem Celular , Núcleo Celular/metabolismo , Quimases/antagonistas & inibidores , Citoplasma/metabolismo , Cães , Endocitose , Isquemia/patologia , Mastócitos/enzimologia , Metaloproteinase 9 da Matriz/metabolismo , Camundongos , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Peptídeo Hidrolases/química , Traumatismo por Reperfusão , Transferrina/metabolismo , Troponina I/sangue
16.
Ther Adv Cardiovasc Dis ; 8(3): 97-118, 2014 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-24692245

RESUMO

OBJECTIVES: Heart failure is typically preceded by myocardial hypertrophy and remodeling, which can be concentric due to pressure overload (PO), or eccentric because of volume overload (VO). The molecular mechanisms that underlie these differing patterns of hypertrophy are distinct and have yet to be fully elucidated. Thus, the goal of this work is to identify novel therapeutic targets for cardiovascular conditions marked by hypertrophy that have previously been resistant to medical treatment, such as a pure VO. METHODS: Concentric or eccentric hypertrophy was induced in rats for 2 weeks with transverse aortic constriction (TAC) or aortocaval fistula (ACF), respectively. Hemodynamic and echocardiographic analysis were used to assess the development of left ventricular (LV) hypertrophy and functional differences between groups. Changes in gene expression were determined by microarray and further characterized with Ingenuity Pathway Analysis. RESULTS: Both models of hypertrophy increased LV mass. Rats with TAC demonstrated concentric LV remodeling while rats with ACF exhibited eccentric LV remodeling. Microarray analysis associated eccentric remodeling with a more extensive alteration of gene expression compared with concentric remodeling. Rats with VO had a marked activation of extracellular matrix genes, promotion of cell cycle genes, downregulation of genes associated with oxidative metabolism, and dysregulation of genes critical to cardiac contractile function. Rats with PO demonstrated similar categorical changes, but with the involvement of fewer individual genes. CONCLUSIONS: Our results indicate that eccentric remodeling is a far more complex process than concentric remodeling. This study highlights the importance of several key biological functions early in the course of VO, including regulation of matrix, metabolism, cell proliferation, and contractile function. Thus, the results of this analysis will inform the ongoing search for new treatments to prevent the progression to heart failure in VO.

17.
Circ Heart Fail ; 7(1): 194-202, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24297688

RESUMO

BACKGROUND: There is currently no therapy proven to attenuate left ventricular (LV) dilatation and dysfunction in volume overload induced by isolated mitral regurgitation (MR). To better understand molecular signatures underlying isolated MR, we performed LV gene expression analyses and overlaid regulated genes into ingenuity pathway analysis in patients with isolated MR. METHODS AND RESULTS: Gene arrays from LV tissue of 35 patients, taken at the time of surgical repair for isolated MR, were compared with 13 normal controls. Cine-MRI was performed in 31 patients before surgery to measure LV function and volume from serial short-axis summation. LV end-diastolic volume was 2-fold (P=0.005) higher in MR patients than in normal controls, and LV ejection fraction was 64±7% (50%-79%) in MR patients. Ingenuity pathway analysis identified significant activation of pathways involved in ß-adrenergic, cAMP, and G-protein-coupled signaling, whereas there was downregulation of pathways associated with complement activation and acute phase response. SERCA2a and phospholamban protein were unchanged in MR versus control left ventricles. However, mRNA and protein levels of the sarcoplasmic reticulum Ca2+ ATPase (SERCA) regulatory protein sarcolipin, which is predominantly expressed in normal atria, were increased 12- and 6-fold, respectively. Immunofluorescence analysis confirmed the absence of sarcolipin in normal left ventricles and its marked upregulation in MR left ventricles. CONCLUSIONS: These results demonstrate alterations in multiple pathways associated with ß-adrenergic signaling and sarcolipin in the left ventricles of patients with isolated MR and LV ejection fraction>50%, suggesting a beneficial role for ß-adrenergic blockade in isolated MR.


Assuntos
Adrenérgicos/metabolismo , Insuficiência da Valva Mitral/metabolismo , Proteínas Musculares/metabolismo , Proteolipídeos/metabolismo , Volume Sistólico/fisiologia , Disfunção Ventricular Esquerda/metabolismo , Adulto , Idoso , Biópsia , Estudos de Casos e Controles , Feminino , Perfilação da Expressão Gênica , Ventrículos do Coração/metabolismo , Ventrículos do Coração/patologia , Humanos , Imageamento por Ressonância Magnética , Masculino , Pessoa de Meia-Idade , Insuficiência da Valva Mitral/fisiopatologia , Proteínas Musculares/genética , Proteolipídeos/genética , Transdução de Sinais/fisiologia , Regulação para Cima , Disfunção Ventricular Esquerda/fisiopatologia
18.
Biochem J ; 455(2): 157-67, 2013 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-23924350

RESUMO

Dysfunctional bioenergetics has emerged as a key feature in many chronic pathologies such as diabetes and cardiovascular disease. This has led to the mitochondrial paradigm in which it has been proposed that mtDNA sequence variation contributes to disease susceptibility. In the present study we show a novel animal model of mtDNA polymorphisms, the MNX (mitochondrial-nuclear exchange) mouse, in which the mtDNA from the C3H/HeN mouse has been inserted on to the C57/BL6 nuclear background and vice versa to test this concept. Our data show a major contribution of the C57/BL6 mtDNA to the susceptibility to the pathological stress of cardiac volume overload which is independent of the nuclear background. Mitochondria harbouring the C57/BL6J mtDNA generate more ROS (reactive oxygen species) and have a higher mitochondrial membrane potential relative to those with C3H/HeN mtDNA, independent of nuclear background. We propose this is the primary mechanism associated with increased bioenergetic dysfunction in response to volume overload. In summary, these studies support the 'mitochondrial paradigm' for the development of disease susceptibility, and show that the mtDNA modulates cellular bioenergetics, mitochondrial ROS generation and susceptibility to cardiac stress.


Assuntos
Volume Cardíaco/genética , DNA Mitocondrial/genética , Mitocôndrias/genética , Animais , Dano ao DNA , DNA Mitocondrial/metabolismo , Metabolismo Energético , Predisposição Genética para Doença , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Microscopia Eletrônica de Transmissão , Mitocôndrias/metabolismo , NADH Desidrogenase/genética , NADH Desidrogenase/metabolismo , Estresse Oxidativo/genética , Espécies Reativas de Oxigênio/metabolismo
19.
J Am Soc Hypertens ; 7(2): 128-36, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23312967

RESUMO

Identification of angiotensin-(1-12) [Ang-(1-12)] in forming angiotensin II (Ang II) by a non-renin dependent mechanism has increased knowledge on the paracrine/autocrine mechanisms regulating cardiac expression of Ang peptides. This study now describes in humans the identity of the enzyme accounting for Ang-(1-12) metabolism in the left ventricular (LV) tissue of normal subjects. Reverse phase HPLC characterized the products of (125)I-Ang-(1-12) metabolism in plasma membranes (PMs) from human LV in the absence and presence of inhibitors for chymase (chymostatin), angiotensin-converting enzyme (ACE) 1 (lisinopril) and 2 (MLN-4760), and neprilysin (SHC39370). In the presence of the inhibitor cocktail, ≥ 98% ± 2% of cardiac (125)I-Ang-(1-12) remained intact, whereas exclusion of chymostatin from the inhibitor cocktail led to significant conversion of Ang-(1-12) into Ang II. In addition, chymase-mediated hydrolysis of (125)I-Ang I was higher compared with Ang-(1-12). Negligible Ang-(1-12) hydrolysis occurred by ACE, ACE2, and neprilysin. A high chymase activity was detected for both (125)I-Ang-(1-12) and (125)I-Ang I substrates. Chymase accounts for the conversion of Ang-(1-12) and Ang I to Ang II in normal human LV. These novel findings expand knowledge of the alternate mechanism by which Ang-(1-12) contributes to the production of cardiac angiotensin peptides.


Assuntos
Angiotensinas/metabolismo , Quimases/metabolismo , Ventrículos do Coração/metabolismo , Adulto , Análise de Variância , Angiotensina II/metabolismo , Inibidores da Enzima Conversora de Angiotensina/metabolismo , Western Blotting , Membrana Celular/metabolismo , Cromatografia Líquida de Alta Pressão , Feminino , Regulação Enzimológica da Expressão Gênica , Ventrículos do Coração/citologia , Ventrículos do Coração/enzimologia , Humanos , Hidrólise , Imidazóis/metabolismo , Imuno-Histoquímica , Radioisótopos do Iodo , Leucina/análogos & derivados , Leucina/metabolismo , Lisinopril/metabolismo , Masculino , Pessoa de Meia-Idade , Neprilisina/metabolismo
20.
PLoS One ; 7(11): e49112, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23185298

RESUMO

Multiple phosphorylation sites of Drp1 have been characterized for their functional importance. However, the functional consequence of GSK3beta-mediated phosphorylation of Drp1 remains unclear. In this report, we pinpointed 11 Serine/Threonine sites spanning from residue 634~736 of the GED domain and robustly confirmed Drp1 Ser693 as a novel GSK3beta phosphorylation site. Our results suggest that GSK3beta-mediated phosphorylation at Ser693 does cause a dramatic decrease of GTPase activity; in contrast, GSK3beta-mediated phosphorylation at Ser693 appears not to affect Drp1 inter-/intra-molecular interactions. After identifying Ser693 as a GSK3beta phosphorylation site, we also determined that K679 is crucial for GSK3beta-binding, which strongly suggests that Drp1 is a novel substrate for GSK3beta. Thereafter, we found that overexpressed S693D, but not S693A mutant, caused an elongated mitochondrial morphology which is similar to that of K38A, S637D and K679A mutants. Interestedly, using H89 and LiCl to inhibit PKA and GSK3beta signaling, respectively, it appears that a portion of the elongated mitochondria switched to a fragmented phenotype. In investigating the biofunctionality of phosphorylation sites within the GED domain, cells overexpressing Drp1 S693D and S637D, but not S693A, showed an acquired resistance to H(2)O(2)-induced mitochondrial fragmentation and ensuing apoptosis, which affected cytochrome c, capase-3, -7, and PARP, but not LC3B, Atg-5, Beclin-1 and Bcl2 expressions. These results also showed that the S693D group is more effective in protecting both non-neuronal and neuronal cells from apoptotic death than the S637D group. Altogether, our data suggest that GSK3beta-mediated phosphorylation at Ser693 of Drp1 may be associated with mitochondrial elongation via down-regulating apoptosis, but not autophagy upon H(2)O(2) insult.


Assuntos
Quinase 3 da Glicogênio Sintase/metabolismo , Mitocôndrias/enzimologia , Mitocôndrias/patologia , Estresse Oxidativo , Sequência de Aminoácidos , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Dinaminas/química , Dinaminas/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Glicogênio Sintase Quinase 3 beta , Células HEK293 , Células HeLa , Humanos , Peróxido de Hidrogênio/farmacologia , Hidrólise/efeitos dos fármacos , Lisina/metabolismo , Mitocôndrias/efeitos dos fármacos , Dinâmica Mitocondrial/efeitos dos fármacos , Modelos Biológicos , Dados de Sequência Molecular , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Fosfosserina/metabolismo , Ligação Proteica/efeitos dos fármacos , Estrutura Terciária de Proteína , Transporte Proteico/efeitos dos fármacos , Técnicas do Sistema de Duplo-Híbrido
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