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1.
Cancer Prev Res (Phila) ; 10(2): 142-152, 2017 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-27998883

RESUMO

The mechanism by which aspirin consumption is linked to significant reductions in the incidence of multiple forms of cancer and metastatic spread to distant tissues, resulting in increased cancer patient survival is not well understood. In this study, using colon cancer as an example, we provide both in vitro (cell culture) and in vivo (chemically induced mouse model of colon cancer) evidence that this profound antineoplastic action may be associated with aspirin's ability to irreversibly inhibit COX-1-mediated platelet activation, thereby blocking platelet-cancer cell interactions, which promote cancer cell number and invasive potential. This process may be driven by platelet-induced epithelial-mesenchymal transition (EMT), as assessed using confocal microscopy, based upon changes in cell morphology, growth characteristics and fibronectin expression, and biochemical/molecular analysis by measuring changes in the expression of the EMT markers; vimentin, ß-catenin, and SNAIL. We also provide evidence that a novel, gastrointestinal-safe phosphatidylcholine (PC)-associated aspirin, PL2200 Aspirin, possesses the same or more pronounced actions versus unmodified aspirin with regard to antiplatelet effects (in vitro: reducing platelet activation as determined by measuring the release of thromboxane and VEGF in culture medium; in vivo: inhibiting platelet number/activation and extravasation into tumor tissue) and chemoprevention (in vitro: inhibiting colonic cell growth and invasive activity; in vivo: inhibiting colonic dysplasia, inflammation, and tumor mass). These results suggest that aspirin's chemopreventive effects may be due, in part, to the drug blocking the proneoplastic action of platelets, and the potential use of Aspirin-PC/PL2200 as an effective and safer chemopreventive agent for colorectal cancer and possibly other cancers. Cancer Prev Res; 10(2); 142-52. ©2016 AACR.


Assuntos
Aspirina/farmacologia , Neoplasias do Colo/patologia , Ciclo-Oxigenase 1/metabolismo , Inibidores de Ciclo-Oxigenase/farmacologia , Ativação Plaquetária/efeitos dos fármacos , Animais , Plaquetas/efeitos dos fármacos , Plaquetas/enzimologia , Linhagem Celular Tumoral , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Humanos , Camundongos , Camundongos Endogâmicos BALB C
2.
Open Neurol J ; 10: 67-76, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27651844

RESUMO

BACKGROUND: Specific factors in Parkinson's disease have become targets as to their protective and degenerative effects. We have demonstrated that cytokines and PD-CSF detrimentally affect microglia and astrocyte growth. While glial cell-derived neurotrophic factor (GDNF) has been recognized as a possible neuron-rescue agent, nitric oxide synthase (NOS) has been implicated in neurodegenerative processes. OBJECTIVE: To demonstrate that glial cell activation, cytokine production, and NOS induction, play an intimate role in the loss of dopaminergic signaling, via mechanisms that are a result of inflammation and inflammatory stimuli. METHODS: Study animals were sacrificed following endotoxin treatment and tissue sections were harvested and probed for GDNF and NOS isomers by fluorescence deconvolution microscopy. Fluorescence was mapped and quantified for each probe. RESULTS: An immune cell influx into 'vulnerable' areas of the brain was seen, and three NOS isomers, inducible (iNOS), neuronal (nNOS) and endothelial (eNOS), were synthesized in the brains, a finding which suggests that each isomer has a role in neurodegeneration. eNOS was found associated with blood vessels, while iNOS was associated with glial and matrix cells and nNOS was located with both glia and neurons. Following endotoxin treatment, serum levels of nitric oxide were higher at 6-8 hours, while tissue levels of NOS were elevated for much longer. Thus, induction of NOS occurred earlier than the induction of GDNF. CONCLUSION: Our findings suggest that the protective abilities of GDNF to combat neural destruction are not available rapidly enough, and do not remain at sufficiently high levels long enough to assert its protective effects. (250).

3.
J Interferon Cytokine Res ; 33(7): 376-83, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23600861

RESUMO

This study examined inflammatory cell and cytokine production in brain tissue from a lipopolysaccharide (LPS)-treated rat model that mimics many of the neuropathologic changes associated with neurodegenerative diseases We also monitored the appearance of a glial cell line-derived neurotrophic factor (GDNF) and circulating nitric oxide (NO) levels, as well as an immune system-associated cells in a selected area of the brain, the olfactory lobe. The studies were based on the hypothesis that LPS treatment stimulates temporal changes within the brain and that these responses include immune cell recruitment, increased tissue levels of immune modulating cytokines and NO, as well as greater glial cell activation resulting in increased production of GDNF. As previously reported by other investigators, our animal model of systemic LPS treatment leads to an increase in the concentrations of circulating cytokines, including TNF-α, IL-Iß, and IL-6, with a maximum response 6 h post LPS administration. Concomitant with cytokine elevations, circulating NO levels were elevated for several hours post LPS administration. The brain content of the GDNF was also elevated over a similar time frame. Lymphocytes, neutrophils, macrophages, plasma cells, and cytokines were all seen in various areas of LPS-treated brains, often around blood vessels associated with the meninges, with these localizations possibly indicating involvement of both the blood-brain and blood-cerebral spinal fluid barriers in these inflammatory episodes. Our results suggest an involvement of both the peripheral and the central nervous system immune components in response to inflammation and inflammatory episodes. This leads us to propose that inflammation initiates an immune response by activating both microglia and astrocytes and that the presence of continuing and increasing proinflammatory mechanisms results in a situation, where cellular protective mechanisms are overcome and the more susceptible cells enter into cell death pathways, initiating a train of events that is a major part of neurodegeneration.


Assuntos
Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Inflamação/imunologia , Leucócitos/imunologia , Doenças Neurodegenerativas/imunologia , Bulbo Olfatório/metabolismo , Animais , Barreira Hematoencefálica/imunologia , Movimento Celular/imunologia , Células Cultivadas , Citocinas/metabolismo , Modelos Animais de Doenças , Fator Neurotrófico Derivado de Linhagem de Célula Glial/genética , Humanos , Mediadores da Inflamação/metabolismo , Lipopolissacarídeos/imunologia , Masculino , Óxido Nítrico/metabolismo , Bulbo Olfatório/imunologia , Ratos , Ratos Sprague-Dawley
4.
Eplasty ; 11: e8, 2011 Feb 23.
Artigo em Inglês | MEDLINE | ID: mdl-21369366

RESUMO

OBJECTIVE: To determine alterations in quantities and distributions of natural antimicrobials following ischemia-reperfusion injury. We hypothesized that these compounds would be upregulated in areas of small intestine where changes in permeability and cellular disruption were likely and where protective mechanisms would be initiated. METHODS: Rats with ischemia-reperfusion underwent superior mesenteric artery clamping and reperfusion. Shams were subjected to laparotomy but no clamping. Ileum and jejunum were harvested and sectioned, and subjected to fluorescence deconvolution microscopy for determinations of content and localization of rat beta defensins, 1, 2, 3; rat neutrophil protein-1; and cathelicidin LL-37. Modeling was performed to determine cellular location of antimicrobials. RESULTS: Ischemia-reperfusion increased neutrophil defensin alpha (RNP-1) in jejunum; rat beta defensin 1 was increased 2-fold in ileal mucosa and slightly reduced in jejunal mucosa; rat beta defensin 2 was reduced by ischemia-reperfusion in ileum, but slightly increased in jejunum; rat beta defensin 3 was concentrated in the muscularis externa and myenteric plexus of the jejunum; ischemia-reperfusion did not alter cathelicidin LL-37 content in the small intestine, although a greater concentration was seen in jejunum compared with ileum. CONCLUSION: Ischemia-reperfusion injury caused changes in antimicrobial content in defined areas, and these different regulations might reflect the specific roles of jejunum versus ileum.

5.
ISRN Cardiol ; 2011: 831062, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-22347659

RESUMO

Purpose. To provide an ovine model of ventricular remodeling and reverse remodeling by creating congestive heart failure (CHF) and then treating it by implanting a left ventricular assist device (LVAD). Methods. We induced volume-overload heart failure in 2 sheep; 20 weeks later, we implanted an LVAD and assessed recovery 11 weeks thereafter. We examined changes in histologic and hemodynamic data and levels of cellular markers of CHF. Results. After CHF induction, we found increases in LV end-diastolic pressure, LV systolic and diastolic dimensions, wall thickness, left atrial diameter, and atrial natriuretic protein (ANP) and endothelin-1 (ET-1) levels; ß-adrenergic receptor (BAR) and dystrophin expression decreased markedly. Biopsies confirmed LV remodeling. After LVAD support, LV systolic and diastolic dimensions, wall thickness, and mass, and ANP and ET-1 levels decreased. Histopathologic and hemodynamic markers improved, and BAR and dystrophin expression normalized. Conclusions. We describe a successful sheep model for ventricular and reverse remodeling.

6.
BMC Neurosci ; 11: 151, 2010 Nov 29.
Artigo em Inglês | MEDLINE | ID: mdl-21114836

RESUMO

BACKGROUND: Excessive and abnormal accumulation of alpha-synuclein (α-synuclein) is a factor contributing to pathogenic cell death in Parkinson's disease. The purpose of this study, based on earlier observations of Parkinson's disease cerebrospinal fluid (PD-CSF) initiated cell death, was to determine the effects of CSF from PD patients on the functionally different microglia and astrocyte glial cell lines. Microglia cells from human glioblastoma and astrocytes from fetal brain tissue were cultured, grown to confluence, treated with fixed concentrations of PD-CSF, non-PD disease control CSF, or control no-CSF medium, then photographed and fluorescently probed for α-synuclein content by deconvolution fluorescence microscopy. Outcome measures included manually counted cell growth patterns from day 1-8; α-synuclein density and distribution by antibody tagged 3D model stacked deconvoluted fluorescent imaging. RESULTS: After PD-CSF treatment, microglia growth was reduced extensively, and a non-confluent pattern with morphological changes developed, that was not evident in disease control CSF and no-CSF treated cultures. Astrocyte growth rates were similarly reduced by exposure to PD-CSF, but morphological changes were not consistently noted. PD-CSF treated microglia showed a significant increase in α-synuclein content by day 4 compared to other treatments (p ≤ 0.02). In microglia only, α-synuclein aggregated and redistributed to peri-nuclear locations. CONCLUSIONS: Cultured microglia and astrocytes are differentially affected by PD-CSF exposure compared to non-PD-CSF controls. PD-CSF dramatically impacts microglia cell growth, morphology, and α-synuclein deposition compared to astrocytes, supporting the hypothesis of cell specific susceptibility to PD-CSF toxicity.


Assuntos
Astrócitos/patologia , Proteínas do Líquido Cefalorraquidiano/efeitos adversos , Microglia/patologia , Doença de Parkinson/líquido cefalorraquidiano , Astrócitos/fisiologia , Morte Celular/fisiologia , Diferenciação Celular/fisiologia , Linhagem Celular Tumoral , Proliferação de Células , Forma Celular/fisiologia , Células Cultivadas , Humanos , Corpos de Lewy/metabolismo , Microglia/fisiologia , Doença de Parkinson/imunologia , Doença de Parkinson/metabolismo , alfa-Sinucleína/fisiologia
7.
Parkinsonism Relat Disord ; 16(10): 645-9, 2010 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-20846895

RESUMO

OBJECTIVE: The purpose of this study was to quantify volumes of specific subcortical gray matter nuclei implicated in Parkinson's disease (PD) as a preliminary step for identifying a non-invasive clinical biomarker for PD. We hypothesized that REM sleep behavior disorder (RBD) patients, at risk for developing PD, will demonstrate a pattern of neuronal degeneration reflected in reduced striatal volumes on T1-weighted MRI. METHODS: We compared measures of RBD patients confirmed by polysomnography (PSG) with groups of age/gender-matched Control subjects and early PD (EPD) patients (Hoehn & Yahr < 2). Clinical measurements included the Unified Parkinson's disease Rating Scales (UPDRS), timed gait and finger tapping tasks, the Parkinson's Disease Questionnaire (PDQ-39), and a time-synchronized video recorded single-night PSG. Volumetric measurements were derived from high-resolution T1-weighted 3 T MRI images. RESULTS: The matched Control and EPD groups were statistically similar to the RBD group in age, gender, handedness, and total brain volumes. The RBD group had smaller bilateral putamen volumes (both raw and normalized by brain tissue volume), in addition to some clinical impairment on the UPDRS and PDQ-39. CONCLUSIONS: Reduced putamen volumes may be a structural marker for RBD and reflect a pattern of neurodegeneration that predicts the development of PD.


Assuntos
Putamen/patologia , Transtorno do Comportamento do Sono REM/patologia , Encéfalo/patologia , Núcleo Caudado/patologia , Feminino , Humanos , Processamento de Imagem Assistida por Computador , Corpos de Lewy/patologia , Imageamento por Ressonância Magnética , Masculino , Pessoa de Meia-Idade , Doença de Parkinson/etiologia , Doença de Parkinson/fisiopatologia , Polissonografia , Transtorno do Comportamento do Sono REM/diagnóstico , Risco
8.
Arch Pathol Lab Med ; 134(8): 1188-92, 2010 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-20670141

RESUMO

CONTEXT: Hawthorn is an herb indicated for treating cardiac illness. Because a patient taking digoxin may also take hawthorn, we investigated potential interference of hawthorn in serum digoxin measurements using immunoassays as well as pharmacodynamic interaction between hawthorn and digoxin. Hawthorn contains alkaloids that are structurally similar to digoxin and may interfere with serum digoxin measurement using immunoassays. In addition, hawthorn has cardioactive properties similar to digoxin. OBJECTIVE: To study potential pharmacodynamic interaction between hawthorn and digoxin. DESIGN: The effects of hawthorn extract on serum digoxin measurements using Digoxin III (Abbott Laboratories, Abbott Park, Illinois) and the Tina-Quant digoxin assay (Roche Diagnostics, Indianapolis, Indiana) were investigated using 2 different brands of extract. To study the pharmacodynamic interaction between hawthorn and digoxin, we used an isolated adult rat cardiomyocyte system, measuring calcium transients by real-time fluorescence spectrophotometry. RESULTS: Hawthorn interfered only with the Digoxin III immunoassay but had no effect on the Tina-Quant assay. Both hawthorn extracts increased intracellular calcium levels, but the lack of additive response with digoxin suggests both may bind to the same site of Na, K adenosine triphosphatase. CONCLUSION: Because of interference of hawthorn with a digoxin immunoassay and pharmacodynamic interaction with digoxin, a patient receiving digoxin should avoid hawthorn.


Assuntos
Crataegus/química , Digoxina/sangue , Digoxina/farmacologia , Miócitos Cardíacos/efeitos dos fármacos , Extratos Vegetais/farmacologia , Compostos de Anilina , Animais , Cálcio/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Células Cultivadas , Contraindicações , Interações Medicamentosas , Imunoensaio , Masculino , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Ratos , Xantenos
9.
Am J Pathol ; 175(4): 1555-63, 2009 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-19762713

RESUMO

Cartilage oligomeric matrix protein (COMP) is a pentameric extracellular protein expressed in cartilage and other musculoskeletal tissues. Mutations in the COMP gene cause pseudoachondroplasia (PSACH), a severe dwarfing condition that has a growth plate chondrocyte pathology. PSACH is characterized by intracellular retention of COMP and other extracellular matrix (ECM) proteins, which form an ordered matrix within large rough endoplasmic reticulum cisternae. This accumulation is cytotoxic and causes premature chondrocyte cell death, thereby depleting chondrocytes needed for normal long bone growth. Research to define the underlying molecular mechanisms of PSACH has been hampered by the lack of a suitable model system. In this study, we achieved robust expression of human mutant (MT) or wild-type (WT) COMP in mice by using a tetracycline-inducible promoter. Normal growth plate distribution of ECM proteins was observed in 1-month-old WT-COMP and C57BL\6 control mice. In contrast, the structure of the MT-COMP growth plate recapitulated the findings of human PSACH growth plate morphology, including (1) retention of ECM proteins, (2) intracellular matrix formation in the rER cisternae, and (3) increased chondrocyte apoptosis. Therefore, we have generated the first mouse model to show extensive intracellular retention of ECM proteins recapitulating the human PSACH disease process at the cellular level.


Assuntos
Proteínas da Matriz Extracelular/metabolismo , Glicoproteínas/metabolismo , Osteocondrodisplasias/patologia , Animais , Apoptose , Proteína de Matriz Oligomérica de Cartilagem , Condrócitos/metabolismo , Condrócitos/patologia , Colágeno Tipo IX/metabolismo , Modelos Animais de Doenças , Retículo Endoplasmático/metabolismo , Proteínas da Matriz Extracelular/química , Glicoproteínas/química , Lâmina de Crescimento/anormalidades , Lâmina de Crescimento/patologia , Humanos , Proteínas Matrilinas , Camundongos , Camundongos Transgênicos , Mutação/genética , Fenótipo , Estrutura Quaternária de Proteína
10.
J Leukoc Biol ; 86(4): 981-8, 2009 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-19602668

RESUMO

Mammalian STC1 decreases the mobility of macrophages and diminishes their response to chemokines. In the current experiments, we sought to determine the impact of STC1 on energy metabolism and superoxide generation in mouse macrophages. STC1 decreases ATP level in macrophages but does not affect the activity of respiratory chain complexes I-IV. STC1 induces the expression of mitochondrial UCP2, diminishing mitochondrial membrane potential and superoxide generation; studies in UCP2 null and gp91phox null macrophages suggest that suppression of superoxide by STC1 is UCP2-dependent yet is gp91phox-independent. Furthermore, STC1 blunts the effects of LPS on superoxide generation in macrophages. Exogenous STC1 is internalized by macrophages within 10 min and localizes to the mitochondria, suggesting a role for circulating and/or tissue-derived STC1 in regulating macrophage function. STC1 induces arrest of the cell cycle at the G1 phase and reduces cell necrosis and apoptosis in serum-starved macrophages. Our data identify STC1 as a key regulator of superoxide generation in macrophages and suggest that STC1 may profoundly affect the immune/inflammatory response.


Assuntos
Glicoproteínas/imunologia , Canais Iônicos/imunologia , Macrófagos Peritoneais/imunologia , Glicoproteínas de Membrana/imunologia , Mitocôndrias/imunologia , Proteínas Mitocondriais/imunologia , NADPH Oxidases/imunologia , Superóxidos/imunologia , Trifosfato de Adenosina/genética , Trifosfato de Adenosina/imunologia , Animais , Apoptose/genética , Apoptose/imunologia , Linhagem Celular , Movimento Celular/genética , Movimento Celular/imunologia , Transporte de Elétrons/imunologia , Fase G1/genética , Fase G1/imunologia , Regulação da Expressão Gênica/genética , Regulação da Expressão Gênica/imunologia , Glicoproteínas/genética , Canais Iônicos/genética , Macrófagos Peritoneais/patologia , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Knockout , Mitocôndrias/genética , Proteínas Mitocondriais/genética , NADPH Oxidase 2 , NADPH Oxidases/genética , Necrose/genética , Necrose/imunologia , Transporte Proteico/genética , Transporte Proteico/imunologia , Fatores de Tempo , Proteína Desacopladora 2
11.
Open Cardiovasc Med J ; 3: 1-7, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-19430572

RESUMO

Neonatal and adult cardiomyocytes were isolated from rat hearts. Some of the adult myocytes were cultured to allow for cell dedifferentiation, a phenomenon thought to mimic cell changes that occur in stressed myocardium, with myocytes regressing to a fetal pattern of metabolism and stellate neonatal shape.Using fluorescence deconvolution microscopy, cells were probed with fluorescent markers and scanned for a number of proteins associated with ion control, calcium movements and cardiac function. Image analysis of deconvoluted image stacks and sequential real-time image recordings of calcium transients of cells were made.All three myocyte groups were predominantly comprised of binucleate cells. Clustering of proteins to a single nucleus was a common observation, suggesting that one nucleus is active in protein synthesis pathways, while the other nucleus assumes a 'dormant' or different role and that cardiomyocytes might be mitotically active even in late development, or specific protein syntheses could be targeted and regulated for reintroduction into the cell cycle.Such possibilities would extend cardiac disease associated stem cell research and therapy options, while producing valuable insights into developmental and death pathways of binucleate cardiomyocytes (word count 183).

12.
Cytokine ; 45(3): 179-83, 2009 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-19157893

RESUMO

Human derived glioblastoma cells were cultured and treated with cytokines interleukin-6 (IL6), tumor necrosis factor alpha (TNF) and interferon-gamma (IFN) and imaged by fluorescence deconvolution microscopy to localize alpha-synuclein, tau and ubiquitin. Exposures were for short (2 h) and prolonged times (up to 96 h), with doses at both low (10 ng/ml), and high (100 ng/ml) concentrations. Further experiments used additive doses up to 200 ng/ml (2 x 100 ng), mimicking a super-infection state. Single, low doses of the cytokines initiated changes in levels of intracellular proteins, but these changes, be they increases or decreases, were not sustained, so we added higher doses of cytokine to the culture medium or fresh aliquots of cytokines over time. Finally, we treated cells with high, single doses of cytokine (200 ng/ml), to try to sustain perturbations of the proteins with cytokines. IFN caused a disruption and reduction of peripheral synuclein, TNF treatment resulted in increased levels of ubiquitin and IL6 disrupted and appeared to fragment tau. Of note, each of the proteins was found in a specific locale, tau being perinuclear, ubiquitin residing in the cytoplasm, and alpha-synuclein occupying the tips of cellular processes, exhibiting the characteristics of an adhesion protein/molecule [Word count=198].


Assuntos
Glioblastoma/metabolismo , Interferon gama/farmacologia , Interleucina-6/farmacologia , Doença de Parkinson/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Ubiquitina/metabolismo , alfa-Sinucleína/metabolismo , Proteínas tau/metabolismo , Células Cultivadas , Humanos , Imageamento Tridimensional , Microscopia de Fluorescência , Ubiquitina/efeitos dos fármacos , alfa-Sinucleína/efeitos dos fármacos , Proteínas tau/efeitos dos fármacos
13.
Eplasty ; 10: e6, 2009 Dec 26.
Artigo em Inglês | MEDLINE | ID: mdl-20076788

RESUMO

OBJECTIVE: The aim of this study was to visualize and localize the sheep antimicrobials, beta-defensins 1, 2, and 3, (SBD-1, SBD-2, SBD-3), sheep neutrophil defensin alpha (SNP-1), and the cathelicidin LL-37 in sheep small intestine after burn injury, our hypothesis being that these compounds would be upregulated in an effort to overcome a compromised endothelial lining. Response to burn injury includes the release of proinflammatory cytokines and systemic immune suppression that, if untreated, can progress to multiple organ failure and death, so protective mechanisms have to be initiated and implemented. METHODS: Tissue sections were probed with antibodies to the antimicrobials and then visualized with fluorescently labeled secondary antibodies and subjected to fluorescence deconvolution microscopy and image reconstruction. RESULTS: In both the sham and burn samples, all the aforementioned antimicrobials were seen in each of the layers of small intestine, the highest concentration being localized to the epithelium. SBD-2, SBD-3, and SNP-1 were upregulated in both enterocytes and Paneth cells, while SNP-1 and LL-37 showed increases in both the inner circular and outer longitudinal muscle layers of the muscularis externa following burn injury. Each of the defensins, except SBD-1, was also seen in between the muscle layers of the externa and while burn caused slight increases of SBD-2, SBD-3, and SNP-1 in this location, LL-37 content was significantly decreased. CONCLUSION: That while each of these human antimicrobials is present in multiple layers of sheep small intestine, SBD-2, SBD-3, SNP-1, and LL-37 are upregulated in the specific layers of the small intestine.

14.
J Med Food ; 11(4): 680-6, 2008 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-19053860

RESUMO

We studied the potential cardiac effects of two alcohol extracts of commercially available hawthorn using rat cardiomyocytes and measuring calcium transients by real-time fluorescence spectrophotometry. One preparation was a blend of hawthorn flowers, leaves, and berries (extract #1), and the other (extract #2) was from a "berries-only" preparation. Fluorescent images and calcium transients were acquired concurrently. Addition of extract #1 resulted in the initiation of robust calcium transients and eventual calcium overload, while addition of extract #2 caused increased calcium sparking, initiation of calcium transients, and an increased beating rate but no calcium overload. To identify the mechanisms of increased calcium influx, adult rat cardiomyocytes were challenged with 10 microM ouabain, a Na(+),K(+)-ATPase inhibitor, and the calcium channel blocker nifedipine. The findings revealed that equal volumes of the two readily available hawthorn preparations demonstrated markedly different effects on isolated adult rat cardiomyocytes, suggesting important implications for patients who are using these preparations to supplement or even replace their prescribed cardiac medications as to which preparation(s) to use, and potential dire consequences, particularly in cardiac patients. Our study indicates that the mechanism of cardiac activity of hawthorn is via the Na(+),K(+)-ATPase and intracellular calcium concentrations are influenced.


Assuntos
Cálcio/metabolismo , Crataegus , Miócitos Cardíacos/efeitos dos fármacos , Fitoterapia , Extratos Vegetais/farmacologia , Animais , Bloqueadores dos Canais de Cálcio/farmacologia , Cardiotônicos/farmacologia , Insuficiência Cardíaca/tratamento farmacológico , Interações Ervas-Drogas , Hipertensão/tratamento farmacológico , Masculino , Miócitos Cardíacos/metabolismo , Nifedipino/farmacologia , Extratos Vegetais/uso terapêutico , Estruturas Vegetais , Ratos , Ratos Sprague-Dawley , ATPase Trocadora de Sódio-Potássio/metabolismo
15.
J Thorac Cardiovasc Surg ; 136(2): 343-51, 2008 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-18692640

RESUMO

OBJECTIVES: The aim of this study was to test the hypothesis that after an acute myocardial infarction, endothelin-1 release with subsequent calcium overload is a mediator of myocardial reperfusion injury, which can be inhibited, in part, by left ventricular unloading immediately before reperfusion. We recently have reported that left ventricular unloading before reperfusion reduces infarct size after acute myocardial infarction. However, the biologic mechanisms of infarct salvage in unloaded hearts subjected to ischemia/reperfusion remain undefined. METHODS: Twelve pigs were subjected to 1 hour of left anterior descending coronary artery occlusion followed by 4 hours of reperfusion. A left ventricular assist device was initiated 15 minutes before reperfusion and maintained during reperfusion (assist device group, n = 6). A control group (n = 6) was subjected to reperfusion alone. Infarct size, endothelin-1 plasma levels, intracellular calcium levels, and apoptosis were analyzed in both groups. RESULTS: At reperfusion, left ventricular unloading significantly decreased left ventricular end-diastolic and end-systolic pressures. Infarct size, expressed as a percentage of zone at risk, was also significantly reduced by 54% in the group with the left ventricular assist device compared with controls. Support with a left ventricular assist device reduced endothelin-1 release from the heart at 15 minutes, 30 minutes, and 1 hour of reperfusion. Myocardial release of endothelin-1 was significantly correlated with infarct size at 15 minutes of reperfusion (r = 0.79; P = .008). Left ventricular unloading caused a significant reduction of calcium overload and of the percentage of apoptotic cells in the ischemic region. CONCLUSION: Our findings suggest that endothelin-1 release and calcium overload are important mediators of reperfusion injury and that they can be significantly reduced by left ventricular unloading before coronary artery reperfusion during myocardial infarction.


Assuntos
Cálcio/metabolismo , Endotelina-1/sangue , Endotelina-1/metabolismo , Infarto do Miocárdio/metabolismo , Reperfusão Miocárdica , Função Ventricular Esquerda , Animais , Apoptose , Circulação Coronária , Hemodinâmica , Marcação In Situ das Extremidades Cortadas , Infarto do Miocárdio/patologia , Infarto do Miocárdio/fisiopatologia , Traumatismo por Reperfusão Miocárdica/metabolismo , Miocárdio/metabolismo , Sus scrofa
16.
J Heart Lung Transplant ; 27(7): 710-7, 2008 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-18582798

RESUMO

BACKGROUND: We hypothesized that not all subtypes of alpha- and beta-adrenoreceptors undergo similar upregulation and redistribution in human myocardium after mechanical unloading with an assist device. METHODS: We obtained core biopsy samples of the left ventricle in 19 patients before and after removal of a Jarvik or Thoratec left ventricular assist device (LVAD) to study the effect of mechanical unloading on the distribution of alpha- and beta-adrenoreceptors. Fresh, embedded tissue sections were incubated with receptor blockers and antibodies before the fluorescent labeling of receptors. Images were obtained by fluorescence deconvolution microscopy, and composite tissue renditions were made from the stacked images. Multiple adrenoreceptor subtypes were studied. RESULTS: We saw a reversal of myocyte hypertrophy in all patients, but the upregulation of receptors was not seen in all post-LVAD tissue samples. Furthermore, we noted receptor relocalization from an initial punctate/clumped pattern to a normal homogeneous distribution in many patients. Significant differences were seen in the distribution of beta(2)- and alpha(1)-receptors and in alpha(1A) subtypes. CONCLUSIONS: In this study we show not only the expected reversal of myocyte hypertrophy and the increase in adrenoreceptors after ventricular unloading, but also the relocalization of specific receptor subtypes.


Assuntos
Insuficiência Cardíaca/patologia , Insuficiência Cardíaca/terapia , Miocárdio/patologia , Receptores Adrenérgicos alfa , Receptores Adrenérgicos beta , Adulto , Biópsia por Agulha , Feminino , Transplante de Coração , Coração Auxiliar , Humanos , Masculino , Pessoa de Meia-Idade
17.
Brain Res ; 1217: 203-12, 2008 Jun 27.
Artigo em Inglês | MEDLINE | ID: mdl-18501880

RESUMO

The purpose of this study was to determine the effects of specific proinflammatory cytokines interleukin-6 (Il-6), interleukin-1beta (Il-1beta), interferon-gamma (IFN), and tumor necrosis factor-alpha (TNFalpha), on content and distribution of alpha-synuclein (alpha-synuclein), tau and ubiquitin in human derived cultured glial cells. Exposure paradigms mimicked acute (2 h), intermediate (18 h) and prolonged time frames (96 h); consisting of single or repeated low doses (10 ng/ml) or high doses (50 ng/ml), consistent with either mild or serious systemic infectious/inflammatory responses. Images of intracellular protein content and distribution were reconstructed from emission patterns generated by fluorescence deconvolution microscopy. Minor alterations were seen in protein content with IFN; Il-1beta decreased alpha-synuclein and tau at 18 and 96 h; TNFalpha inversely reduced alpha-synuclein and increased ubiquitin content. Combinations of Il-1beta and IFN produced a robust increase of alpha-synuclein and tau at 2 h. Consecutive low doses of Il-6 produced only minor increases in alpha-synuclein and ubiquitin after 4 h, whereas a single high dose resulted in major increases for all three proteins over the first 18 h. Protein localization patterns were distinctly different and were altered dependent upon cytokine treatment. A high dose exposure (2 x 50 ng/ml) with Il-6 and IFN demonstrated that protein increases and dispersals could be sustained and that the normal perinuclear tau and peripheral alpha-synuclein patterns were disrupted. These results support the postulate that specific cytokines affect temporal protein changes with concomitant pattern disruptions, possibly reflecting a mechanism of cell dysfunction in Parkinson's degeneration.


Assuntos
Citocinas/metabolismo , Neuroglia/metabolismo , Doença de Parkinson/metabolismo , Ubiquitina/metabolismo , alfa-Sinucleína/metabolismo , Proteínas tau/metabolismo , Células Cultivadas , Humanos , Microscopia de Fluorescência , Neuroglia/química
18.
Burns ; 34(3): 370-5, 2008 May.
Artigo em Inglês | MEDLINE | ID: mdl-17869425

RESUMO

Due to up-regulation of the parathyroid gland calcium-sensing receptor (CaR), burned children have hypocalcemic hypoparathyroidism, and decreased myocardial contractility. Our aim was to localize the CaR in the heart and measure receptor density changes due to burns. Heart and aorta samples from sheep subjected to 40% burn or sham conditions were probed for CaR via fluorescence microscopy. CaR was localized to endocardial endothelium, myocardial microvasculature, and fibroblasts and vessels of the aortic adventitia. CaR was not found in cardiomyocytes or smooth muscle cells. No differences in density of CaR or beta-adrenergic receptors were noted. No differences in CaR distribution were seen in the myocardium or aorta, in contrast to the parathyroid where burn injury up-regulates CaR. We suggest that CaR has a local, tissue-specific role, and functions in vascular calcium sensing for intravascular calcium deposition or regulation of other calcium channels after trauma or burn.


Assuntos
Aorta/metabolismo , Queimaduras/metabolismo , Miocárdio/metabolismo , Receptores de Detecção de Cálcio/metabolismo , Animais , Ovinos
19.
J Burns Wounds ; 7: e3, 2007 Aug 24.
Artigo em Inglês | MEDLINE | ID: mdl-18091980

RESUMO

OBJECTIVE: Human defensins and cathelicidins are a family of cationic antimicrobial peptides (AMPs), which play multiple roles in both innate and adaptive immune systems. They have direct antimicrobial activity against several microorganisms including burn pathogens. The majority of components of innate and adaptive immunity either express naturally occurring defensins or are otherwise chemoattracted or functionally affected by them. They also enhance adaptive immunity and wound healing and alter antibody production. All mechanisms to explain multiple functions of AMPs are not clearly understood. Prior studies to localize defensins in normal and burned skin using deconvolution fluorescence scanning microscopy indicate localization of defensins in the nucleus, perinuclear regions, and cytoplasm. The objective of this study is to further confirm the identification of HBD-1 in the nucleus by deconvolution microscopic studies involving image reconstruction and wire frame modeling. RESULTS: Our study demonstrated the presence of intranuclear HBD-1 in keratinocytes throughout the stratum spinosum by costaining with the nuclear probe DAPI. In addition, HBD-1 sequence does show some homology with known cationic nuclear localization signal sequences. CONCLUSION: To our knowledge, this is the first report to localize HBD-1 in the nuclear region, suggesting a role for this peptide in gene expression and providing new data that may help determine mechanisms of defensin functions.

20.
J Toxicol Environ Health A ; 70(6): 568-74, 2007 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-17365610

RESUMO

Oleanders are common, hardy shrubs that grow throughout the southern United States. They contain cardiotonic steroids formed from cardenolides and bufadienolides, making the plant poisonous to both animals and humans. Aliquots of both commercially available oleander and fresh oleander extracts were prepared. Fresh, rod-like, calcium-tolerant adult rat cardiomyocytes and cultured neonatal cardiomyocytes were isolated and treated with 0-4 ng/ml of both preparations, challenged with verapamil and ouabain, and real-time spectrophotometric calcium transients and images were acquired. A number of effects were observed with the adult cells: (1) intracellular calcium levels were increased in a concentration-dependent manner: (2) reduced calcium transient heights and eventual cessation of beating resulted; and (3) increased sparking intensity led to subsequent beating and eventual calcium overload. In the spontaneously beating cultured neonatal myocytes increased intramyocytic calcium levels were also seen, with retention of this calcium rise leading to overload and, as in the adult myocytes, cessation of beating. These observations demonstrate that oleander extract is markedly potent with respect to the elevation of calcium concentrations in cardiomyocytes, and that the inability of the cardiomyocytes to release the accumulated calcium possibly indicates a role for oleandrin in inhibition of ryanodine receptor calcium release channels, calcium uptake via Na+,K+-ATPase inhibition [EC 3.6.1.3], and/or dysfunction of sarcolemmal calcium release channels.


Assuntos
Envelhecimento/fisiologia , Cálcio/metabolismo , Cardenolídeos/toxicidade , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Animais , Animais Recém-Nascidos , Bloqueadores dos Canais de Cálcio/farmacologia , Células Cultivadas , Masculino , Nerium/química , Extratos Vegetais/química , Extratos Vegetais/farmacologia , Ratos , Ratos Sprague-Dawley , Verapamil/farmacologia
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