RESUMEN
Pseudomonas aeruginosa is a Gram-negative, opportunistic pathogen that causes nosocomial pneumonia, urinary tract infections, and bacteremia. A hallmark of P. aeruginosa pathogenesis is disruption of host cell function by the type III secretion system (T3SS) and its cognate exoenzyme effectors. The T3SS effector ExoU is phospholipase A2 (PLA2) that targets the host cell plasmalemmal membrane to induce cytolysis and is an important virulence factor that mediates immune avoidance. In addition, ExoU has been shown to subvert the host inflammatory response in a noncytolytic manner. In primary bone marrow-derived macrophages (BMDMs), P. aeruginosa infection is sensed by the nucleotide-binding domain containing leucine-rich repeats-like receptor 4 (NLRC4) inflammasome, which triggers caspase-1 activation and inflammation. ExoU transiently inhibits NLRC4 inflammasome-mediated activation of caspase-1 and its downstream target, interleukin 1ß (IL-1ß), to suppress activation of inflammation. In the present study, we sought to identify additional noncytolytic virulence functions for ExoU and discovered an unexpected association between ExoU, host mitochondria, and NLRC4. We show that infection of BMDMs with P. aeruginosa strains expressing ExoU elicited mitochondrial oxidative stress. In addition, mitochondria and mitochondrion-associated membrane fractions enriched from infected cells exhibited evidence of autophagy activation, indicative of damage. The observation that ExoU elicited mitochondrial stress and damage suggested that ExoU may also associate with mitochondria during infection. Indeed, ExoU phospholipase A2 enzymatic activity was present in enriched mitochondria and mitochondrion-associated membrane fractions isolated from P. aeruginosa-infected BMDMs. Intriguingly, enriched mitochondria and mitochondrion-associated membrane fractions isolated from infected Nlrc4 homozygous knockout BMDMs displayed significantly lower levels of ExoU enzyme activity, suggesting that NLRC4 plays a role in the ExoU-mitochondrion association. These observations prompted us to assay enriched mitochondria and mitochondrion-associated membrane fractions for NLRC4, caspase-1, and IL-1ß. NLRC4 and pro-caspase-1 were detected in enriched mitochondria and mitochondrion-associated membrane fractions isolated from noninfected BMDMs, and active caspase-1 and active IL-1ß were detected in response to P. aeruginosa infection. Interestingly, ExoU inhibited mitochondrion-associated caspase-1 and IL-1ß activation. The implications of ExoU-mediated effects on mitochondria and the NLRC4 inflammasome during P. aeruginosa infection are discussed.
Asunto(s)
Infecciones por Pseudomonas , Pseudomonas aeruginosa , Animales , Caspasa 1/metabolismo , Inflamasomas/metabolismo , Inflamación/metabolismo , Macrófagos/metabolismo , Ratones , Fosfolipasas/metabolismo , Pseudomonas aeruginosa/fisiología , Sistemas de Secreción Tipo III/metabolismoRESUMEN
In hypoxia, mitochondria-generated reactive oxygen species not only stimulate accumulation of the transcriptional regulator of hypoxic gene expression, hypoxia inducible factor-1 (Hif-1), but also cause oxidative base modifications in hypoxic response elements (HREs) of hypoxia-inducible genes. When the hypoxia-induced base modifications are suppressed, Hif-1 fails to associate with the HRE of the VEGF promoter, and VEGF mRNA accumulation is blunted. The mechanism linking base modifications to transcription is unknown. Here we determined whether recruitment of base excision DNA repair (BER) enzymes in response to hypoxia-induced promoter modifications was required for transcription complex assembly and VEGF mRNA expression. Using chromatin immunoprecipitation analyses in pulmonary artery endothelial cells, we found that hypoxia-mediated formation of the base oxidation product 8-oxoguanine (8-oxoG) in VEGF HREs was temporally associated with binding of Hif-1α and the BER enzymes 8-oxoguanine glycosylase 1 (Ogg1) and redox effector factor-1 (Ref-1)/apurinic/apyrimidinic endonuclease 1 (Ape1) and introduction of DNA strand breaks. Hif-1α colocalized with HRE sequences harboring Ref-1/Ape1, but not Ogg1. Inhibition of BER by small interfering RNA-mediated reduction in Ogg1 augmented hypoxia-induced 8-oxoG accumulation and attenuated Hif-1α and Ref-1/Ape1 binding to VEGF HRE sequences and blunted VEGF mRNA expression. Chromatin immunoprecipitation-sequence analysis of 8-oxoG distribution in hypoxic pulmonary artery endothelial cells showed that most of the oxidized base was localized to promoters with virtually no overlap between normoxic and hypoxic data sets. Transcription of genes whose promoters lost 8-oxoG during hypoxia was reduced, while those gaining 8-oxoG was elevated. Collectively, these findings suggest that the BER pathway links hypoxia-induced introduction of oxidative DNA modifications in promoters of hypoxia-inducible genes to transcriptional activation.
Asunto(s)
Daño del ADN/genética , Reparación del ADN/genética , Regulación de la Expresión Génica , Regiones Promotoras Genéticas , Factor A de Crecimiento Endotelial Vascular/genética , Animales , Sitios de Unión , Hipoxia de la Célula/genética , Inmunoprecipitación de Cromatina , Células Endoteliales/metabolismo , Guanina/análogos & derivados , Guanina/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Motivos de Nucleótidos , Oxidación-Reducción , Arteria Pulmonar/patología , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Interferente Pequeño/metabolismo , Ratas , Elementos de Respuesta/genética , Factores de Tiempo , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Tetracycline-inducible systems allow for either suppression or induction of transgene expression to facilitate studies of cell physiology. Doxycycline is a preferred inducer for these gene expression systems due to its membrane permeability; however, the heterocyclic structure of doxycycline exhibits fluorogenic properties that can potentially bias measurement of other fluorochromes. Thus the simultaneous use of tetracycline-inducible systems and fluorescent proteins as reporter genes or as intracellular biosensors may lead to potentially confounding results. Herein, using cells which co-express the ratiometric redox sensitive intracellular reporter, roGFP, and a tetracycline-inducible reporter plasmid encoding the reporter gene, mCherry, as a model system, we describe the overlapping intracellular fluorescent signals between doxycycline and commonly used intracellular fluorescent probes. In our cells, the addition of doxycycline to cells caused a dose- and time-dependent increase in cell fluorescence with 405 nm excitation which overlapped with that of the oxidized configuration of roGFP. Incubating cells in concentrations of doxycycline less than 1 µg/mL and removing doxycycline from the media 60 min before performing experiments eliminated fluorescence interference while still maintaining maximal reporter transgene activation.
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Doxiciclina/química , Genes Reporteros , Proteínas Fluorescentes Verdes/química , Animales , Células Cultivadas , Fluorescencia , Expresión Génica , Proteínas Fluorescentes Verdes/genética , Oxidación-Reducción , Ratas , Espectrometría de FluorescenciaRESUMEN
The Gram-negative, opportunistic pathogen Pseudomonas aeruginosa utilizes a type III secretion system to inject exoenzyme effectors into a target host cell. Of the four best-studied exoenzymes, ExoU causes rapid cell damage and death. ExoU is a phospholipase A2 (PLA2) that hydrolyses host cell membranes, and P. aeruginosa strains expressing ExoU are associated with poor outcomes in critically ill patients with pneumonia. While the effects of ExoU on lung epithelial and immune cells are well studied, a role for ExoU in disrupting lung endothelial cell function has only recently emerged. Lung endothelial cells maintain a barrier to fluid and protein flux into tissue and airspaces and regulate inflammation. Herein, we describe a pulmonary microvascular endothelial cell (PMVEC) culture infection model to examine the effects of ExoU. Using characterized P. aeruginosa strains and primary clinical isolates, we show that strains expressing ExoU disrupt PMVEC barrier function by causing substantial PMVEC damage and lysis, in a PLA2-dependent manner. In addition, we show that strains expressing ExoU activate the pro-inflammatory caspase-1, in a PLA2-dependent manner. Considering the important roles for mitochondria and oxidative stress in regulating inflammatory responses, we next examined the effects of ExoU on reactive oxygen species production. Infection of PMVECs with P. aeruginosa strains expressing ExoU triggered a robust oxidative stress compared to strains expressing other exoenzyme effectors. We also provide evidence that, intriguingly, ExoU PLA2 activity was detectable in mitochondria and mitochondria-associated membrane fractions isolated from P. aeruginosa-infected PMVECs. Interestingly, ExoU-mediated activation of caspase-1 was partially inhibited by reactive oxygen species scavengers. Together, these data suggest ExoU exerts pleiotropic effects on PMVEC function during P. aeruginosa infection that may inhibit endothelial barrier and inflammatory functions.
Asunto(s)
Proteínas Bacterianas/toxicidad , Caspasa 1/efectos de los fármacos , Muerte Celular/efectos de los fármacos , Enfermedad Hepática Inducida por Sustancias y Drogas/fisiopatología , Células Endoteliales/efectos de los fármacos , Infecciones por Pseudomonas/fisiopatología , Pseudomonas aeruginosa/genética , Caspasa 1/metabolismo , Variación Genética , Genotipo , Humanos , Inflamación/inducido químicamente , Inflamación/fisiopatología , Infecciones por Pseudomonas/genéticaRESUMEN
Pulmonary microvascular endothelial cells possess both highly proliferative and angiogenic capacities, yet it is unclear how these cells sustain the metabolic requirements essential for such growth. Rapidly proliferating cells rely on aerobic glycolysis to sustain growth, which is characterized by glucose consumption, glucose fermentation to lactate, and lactic acidosis, all in the presence of sufficient oxygen concentrations. Lactate dehydrogenase A converts pyruvate to lactate necessary to sustain rapid flux through glycolysis. We therefore tested the hypothesis that pulmonary microvascular endothelial cells express lactate dehydrogenase A necessary to utilize aerobic glycolysis and support their growth. Pulmonary microvascular endothelial cell (PMVEC) growth curves were conducted over a 7-day period. PMVECs consumed glucose, converted glucose into lactate, and acidified the media. Restricting extracellular glucose abolished the lactic acidosis and reduced PMVEC growth, as did replacing glucose with galactose. In contrast, slow-growing pulmonary artery endothelial cells (PAECs) minimally consumed glucose and did not develop a lactic acidosis throughout the growth curve. Oxygen consumption was twofold higher in PAECs than in PMVECs, yet total cellular ATP concentrations were twofold higher in PMVECs. Glucose transporter 1, hexokinase-2, and lactate dehydrogenase A were all upregulated in PMVECs compared with their macrovascular counterparts. Inhibiting lactate dehydrogenase A activity and expression prevented lactic acidosis and reduced PMVEC growth. Thus PMVECs utilize aerobic glycolysis to sustain their rapid growth rates, which is dependent on lactate dehydrogenase A.
Asunto(s)
Proliferación Celular , Endotelio Vascular/citología , Glucólisis , L-Lactato Deshidrogenasa/fisiología , Oxígeno/metabolismo , Arteria Pulmonar/citología , Adenosina Trifosfato/metabolismo , Animales , Western Blotting , Antígenos CD40/fisiología , Endotelio Vascular/enzimología , Glucosa/metabolismo , Isoenzimas/fisiología , Lactato Deshidrogenasa 5 , Ácido Láctico/metabolismo , Masculino , Potencial de la Membrana Mitocondrial , Consumo de Oxígeno , Arteria Pulmonar/enzimología , ARN Mensajero/genética , ARN Interferente Pequeño/farmacología , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Pulmonary vascular endothelial cells express a variety of ion channels that mediate Ca(2+) influx in response to diverse environmental stimuli. However, it is not clear whether Ca(2+) influx from discrete ion channels is functionally coupled to specific outcomes. Thus we conducted a systematic study in mouse lung to address whether the alpha(1G) T-type Ca(2+) channel and the transient receptor potential channel TRPV4 have discrete functional roles in pulmonary capillary endothelium. We used real-time fluorescence imaging for endothelial cytosolic Ca(2+), immunohistochemistry to probe for surface expression of P-selectin, and the filtration coefficient to specifically measure lung endothelial permeability. We demonstrate that membrane depolarization via exposure of pulmonary vascular endothelium to a high-K(+) perfusate induces Ca(2+) entry into alveolar septal endothelial cells and exclusively leads to the surface expression of P-selectin. In contrast, Ca(2+) entry in septal endothelium evoked by the selective TRPV4 activator 4alpha-phorbol-12,13-didecanoate (4alpha-PDD) specifically increases lung endothelial permeability without effect on P-selectin expression. Pharmacological blockade or knockout of alpha(1G) abolishes depolarization-induced Ca(2+) entry and surface expression of P-selectin but does not prevent 4alpha-PDD-activated Ca(2+) entry and the resultant increase in permeability. Conversely, blockade or knockout of TRPV4 specifically abolishes 4alpha-PDD-activated Ca(2+) entry and the increase in permeability, while not impacting depolarization-induced Ca(2+) entry and surface expression of P-selectin. We conclude that in alveolar septal capillaries Ca(2+) entry through alpha(1G) and TRPV4 channels differentially and specifically regulates the transition of endothelial procoagulant phenotype and barrier integrity, respectively.
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Canales de Calcio Tipo T/metabolismo , Calcio/metabolismo , Endotelio Vascular/metabolismo , Selectina-P/metabolismo , Canales Catiónicos TRPV/metabolismo , Animales , Señalización del Calcio , Carcinógenos/farmacología , Endotelio Vascular/citología , Endotelio Vascular/efectos de los fármacos , Técnicas para Inmunoenzimas , Ratones , Ratones Noqueados , Ésteres del Forbol/farmacología , Arteria Pulmonar/metabolismoRESUMEN
Bending and flexing of DNA may contribute to transcriptional regulation. Because hypoxia and other physiological signals induce formation of an abasic site at a key base within the hypoxic response element (HRE) of the vascular endothelial growth factor (VEGF) gene (FASEB J. 19, 387-394, 2005) and because abasic sites can introduce flexibility in model DNA sequences, in the present study we used a fluorescence resonance energy transfer-based reporter system to assess topological changes in a wild-type (WT) sequence of the HRE of the rat VEGF gene and in a sequence harboring a single abasic site mimicking the effect of hypoxia. Binding of the hypoxia-inducible transcriptional complex present in hypoxic pulmonary artery endothelial cell nuclear extract to the WT sequence failed to alter sequence topology whereas nuclear protein binding to the modified HRE engendered considerable sequence flexibility. Topological effects of nuclear proteins on the modified VEGF HRE were dependent on the transcription factor hypoxia-inducible factor-1 and on formation of a single-strand break at the abasic site mediated by the coactivator, Ref-1/Ape1. These observations suggest that oxidative base modifications in the VEGF HRE evoked by physiological signals could be a precursor to single-strand break formation that has an impact on gene expression by modulating sequence flexibility.
Asunto(s)
Proteínas Nucleares/fisiología , Regiones Promotoras Genéticas , Factor A de Crecimiento Endotelial Vascular/genética , Animales , Secuencia de Bases , Células Cultivadas , ADN/metabolismo , Sondas de ADN , Transferencia Resonante de Energía de Fluorescencia , Colorantes Fluorescentes , RatasRESUMEN
Arrest of circulating tumor cells in distant organs is required for hematogenous metastasis, but the tumor cell surface molecules responsible have not been identified. Here, we show that the tumor cell alpha3beta1 integrin makes an important contribution to arrest in the lung and to early colony formation. These analyses indicated that pulmonary arrest does not occur merely due to size restriction, and raised the question of how the tumor cell alpha3beta1 integrin contacts its best-defined ligand, laminin (LN)-5, a basement membrane (BM) component. Further analyses revealed that LN-5 is available to the tumor cell in preexisting patches of exposed BM in the pulmonary vasculature. The early arrest of tumor cells in the pulmonary vasculature through interaction of alpha3beta1 integrin with LN-5 in exposed BM provides both a molecular and a structural basis for cell arrest during pulmonary metastasis.
Asunto(s)
Vasos Sanguíneos/metabolismo , Moléculas de Adhesión Celular/metabolismo , Integrina alfa3/metabolismo , Integrina beta1/metabolismo , Pulmón/irrigación sanguínea , Pulmón/patología , Metástasis de la Neoplasia , Neoplasias/metabolismo , Animales , Anticuerpos/metabolismo , Membrana Basal/metabolismo , Membrana Basal/ultraestructura , Vasos Sanguíneos/anatomía & histología , Adhesión Celular , Línea Celular Tumoral , Humanos , Integrina alfa3/inmunología , Ligandos , Pulmón/metabolismo , Ratones , Invasividad Neoplásica , Ratas , KalininaRESUMEN
High vascular pressure targets the lung septal network, causing acute lung injury. While calcium entry in septal endothelium has been implicated, the channel involved is not known. This study tested the hypothesis that the vanilloid transient receptor potential channel, TRPV4, is a critical participant in the permeability response to high vascular pressure. Isolated lungs from TRPV4(+/+) or TRPV4(-/-) mice were studied at baseline or during high pressure challenge. Permeability was assessed via the filtration coefficient. Endothelial calcium transients were assessed using epifluorescence microscopy of the lung subpleural network. Light microscopy and point counting were used to determine the alveolar fluid volume fraction, a measure of alveolar flooding. Baseline permeability, calcium intensity, and alveolar flooding were no different in TRPV4(+/+) versus TRPV4(-/-) lungs. In TRPV4(+/+) lungs, the high pressure-induced permeability response was significantly attenuated by low calcium perfusate, the TRPV antagonist ruthenium red, the phospholipase A(2) inhibitor methyl arachidonyl fluorophosphonate, or the P450 epoxygenase inhibitor propargyloxyphenyl hexanoic acid. Similarly, the high pressure-induced calcium transient in TRPV4(+/+) lungs was attenuated with ruthenium red or the epoxygenase inhibitor. High vascular pressure increased the alveolar fluid volume fraction compared with control. In lungs from TRPV4(-/-) mice, permeability, calcium intensity, and alveolar fluid volume fraction were not increased. These data support a role for P450-derived epoxyeicosatrienoic acid-dependent regulation of calcium entry via TRPV4 in the permeability response to high vascular pressure.
Asunto(s)
Sistema Enzimático del Citocromo P-450/metabolismo , Hipertensión/complicaciones , Activación del Canal Iónico , Enfermedades Pulmonares/enzimología , Enfermedades Pulmonares/etiología , Canales Catiónicos TRPV/metabolismo , Animales , Ácido Araquidónico/metabolismo , Señalización del Calcio , Endotelio/enzimología , Endotelio/patología , Femenino , Técnicas In Vitro , Masculino , Ratones , Perfusión , Permeabilidad , Pleura/irrigación sanguínea , Pleura/metabolismo , Pleura/patología , Alveolos Pulmonares/metabolismo , Alveolos Pulmonares/patología , TemperaturaRESUMEN
BACKGROUND: The modulation of gap junctional communication between tumor cells and between tumor and vascular endothelial cells during tumorigenesis and metastasis is complex. The notion of a role for loss of gap junctional intercellular communication in tumorigenesis and metastasis has been controversial. While some of the stages of tumorigenesis and metastasis, such as uncontrolled cell division and cellular detachment, would necessitate the loss of intercellular junctions, other stages, such as intravasation, endothelial attachment, and vascularization, likely require increased cell-cell contact. We hypothesized that, in this multi-stage scheme, connexin-43 is centrally involved as a cell adhesion molecule mediating metastatic tumor attachment to the pulmonary endothelium. METHODS: Tumor cell attachment to pulmonary vasculature, tumor growth, and connexin-43 expression was studied in metastatic lung tumor sections obtained after tail-vein injection into nude mice of syngeneic breast cancer cell lines, overexpressing wild type connexin-43 or dominant-negatively mutated connexin-43 proteins. High-resolution immunofluorescence microscopy and Western blot analysis was performed using a connexin-43 monoclonal antibody. Calcein Orange Red AM dye transfer by fluorescence imaging was used to evaluate the gap junction function. RESULTS: Adhesion of breast cancer cells to the pulmonary endothelium increased with cancer cells overexpressing connexin-43 and markedly decreased with cells expressing dominant-negative connexin-43. Upregulation of connexin-43 was observed in tumor cell-endothelial cell contact areas in vitro and in vivo, and in areas of intratumor blood vessels and in micrometastatic foci. CONCLUSION: Connexin-43 facilitates metastatic 'homing' by increasing adhesion of cancer cells to the lung endothelial cells. The marked upregulation of connexin-43 in tumor cell-endothelial cell contact areas, whether in preexisting 'homing' vessels or in newly formed tumor vessels, suggests that connexin-43 can serve as a potential marker of micrometastases and tumor vasculature and that it may play a role in the early incorporation of endothelial cells into small tumors as seeds for vasculogenesis.
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Conexina 43/genética , Neoplasias Pulmonares/secundario , Neoplasias Mamarias Experimentales/patología , Neoplasias Glandulares y Epiteliales/secundario , Neovascularización Patológica/genética , Neovascularización Patológica/patología , Animales , Adhesión Celular/genética , Línea Celular Tumoral , Conexina 43/biosíntesis , Conexina 43/sangre , Endotelio Vascular/metabolismo , Endotelio Vascular/patología , Femenino , Uniones Comunicantes/genética , Regulación Neoplásica de la Expresión Génica , Pulmón/patología , Neoplasias Pulmonares/irrigación sanguínea , Neoplasias Mamarias Experimentales/irrigación sanguínea , Ratones , Ratones Desnudos , Mutación Missense , Trasplante de Neoplasias , Neoplasias Glandulares y Epiteliales/irrigación sanguínea , Neovascularización Patológica/sangre , Ratas , TransfecciónRESUMEN
INTRODUCTION: Activated leukocyte cell adhesion molecule (ALCAM) (CD166) is an immunoglobulin molecule that has been implicated in cell migration. The present study examined the expression of ALCAM in human breast cancer and assessed its prognostic value. METHODS: The immunohistochemical distribution and location of ALCAM was assessed in normal breast tissue and carcinoma. The levels of ALCAM transcripts in frozen tissue (normal breast, n = 32; breast cancer, n = 120) were determined using real-time quantitative PCR. The results were then analyzed in relation to clinical data including the tumor type, the grade, the nodal involvement, distant metastases, the tumor, node, metastasis (TNM) stage, the Nottingham Prognostic Index (NPI), and survival over a 6-year follow-up period. RESULTS: Immunohistochemical staining on tissue sections in ducts/acini in normal breast and in breast carcinoma was ALCAM-positive. Differences in the number of ALCAM transcripts were found in different types of breast cancer. The level of ALCAM transcripts was lower (P = 0.05) in tumors from patients who had metastases to regional lymph nodes compared with those patients without, in higher grade tumors compared with Grade 1 tumors (P < 0.01), and in TNM Stage 3 tumors compared with TNM Stage 1 tumors (P < 0.01). Tumors from patients with poor prognosis (with NPI > 5.4) had significantly lower levels (P = 0.014) of ALCAM transcripts compared with patients with good prognosis (with NPI < 3.4), and tumors from patients with local recurrence had significantly lower levels than those patients without local recurrence or metastases (P = 0.04). Notably, tumors from patients who died of breast cancer had significantly lower levels of ALCAM transcripts (P = 0.0041) than those with primary tumors but no metastatic disease or local recurrence. Patients with low levels of ALCAM transcripts had significantly (P = 0.009) more incidents (metastasis, recurrence, death) compared with patients with primary breast tumors with high levels of ALCAM transcripts. CONCLUSIONS: In the present panel of breast cancer specimens, decreased levels of ALCAM correlated with the nodal involvement, the grade, the TNM stage, the NPI, and the clinical outcome (local recurrence and death). The data suggest that decreased ALCAM expression is of clinical significance in breast cancer, and that reduced expression indicates a more aggressive phenotype and poor prognosis.
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Molécula de Adhesión Celular del Leucocito Activado/metabolismo , Neoplasias de la Mama/metabolismo , Mama/metabolismo , Neoplasias de la Mama/mortalidad , Neoplasias de la Mama/patología , Humanos , Inmunohistoquímica , Metástasis de la Neoplasia , Pronóstico , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de SupervivenciaRESUMEN
We have shown previously that ischemia results in reactive oxygen species production by lung endothelium that occurs within 3-5 s after flow cessation and is followed by lipid peroxidation at 15-30 min as determined by assay of thiobarbituric acid-reactive substances, conjugated dienes, and protein carbonyls in lung homogenate. The present study evaluated membrane lipid peroxidation in isolated, ventilated rat lungs using a fluorescence imaging method that permits continuous observation of pulmonary subpleural microvascular endothelial cells in situ. Diphenyl-1-pyrenylphosphine (DPPP), a fluorescent probe which localizes in the plasma membrane and shows increased fluorescence emission after its oxidation by lipid hydroperoxides, was used for detection of membrane lipid peroxidation. Compared to continuously perfused control lungs, endothelial cell DPPP fluorescence increased significantly within 1 min of ischemia (i.e., flow cessation); these changes were prevented by pretreatment with 0.5 mM alpha-tocopherol succinate (vitamin E) added to the perfusate. Increased DPPP fluorescence was confirmed by spectrofluorometry of lipid extracts of lung homogenates. These data indicate that DPPP can be used for the real-time detection of lipid peroxidation in an intact organ. Ischemia results in peroxidation of the pulmonary microvascular endothelial cell membrane and this insult can be detected as early as 1 min after the onset of ischemia compatible with a radical-mediated process.
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Membrana Celular/química , Membranas Intracelulares/química , Isquemia/metabolismo , Pulmón/metabolismo , Animales , Antioxidantes/farmacología , Membrana Celular/metabolismo , Endotelio Vascular , Fluorescencia , Técnica del Anticuerpo Fluorescente , Colorantes Fluorescentes , Hipoxia/metabolismo , Técnicas para Inmunoenzimas , Membranas Intracelulares/metabolismo , Peroxidación de Lípido/efectos de los fármacos , Peróxidos Lipídicos/metabolismo , Pulmón/enzimología , Masculino , Compuestos Organofosforados , Oxidantes/farmacología , Peroxidasa/metabolismo , Pirenos , Ratas , Ratas Sprague-Dawley , Vitamina E/farmacologíaRESUMEN
The presence of metastases indicates an ominous prognosis in patients with malignancies, yet the factors that distinguish metastatic from non-metastatic tumors remain poorly understood. Here we pursued the hypothesis that apoptosis in vivo would distinguish metastatic cells from non-metastatic cells and developed a novel method for observation of apoptosis induction in living cells. One hour after the infusion of metastatic or non-metastatic human melanoma or transformed rat embryo fibroblasts, arrest of tumor cells in the pulmonary vasculature was equivalent. In order to demonstrate the induction of apoptosis in living cells, we observed the translocation of cytoplasmic BAD-GFP fusion proteins to the mitochondria during apoptosis. Microscopic observation of the tumor cells transfected with BAD-GFP in isolated lung preparations after intravenous injection into nu/nu mice revealed translocation of BAD-GFP in many more of the arrested, non-metastatic melanoma or transformed rat embryo cells over 4-24 h than of the metastatic cells. TUNEL staining confirmed enhanced apoptosis by non-metastatic tumor cells after injection in vivo. Metastatic melanoma cells or metastatic embryo fibroblasts were better able to negotiate the barrier of survival in the circulation after pulmonary arrest than non-metastatic cells confirming the hypothesis that susceptibility to apoptosis after arrest in the pulmonary vasculature distinguishes metastatic from non-metastatic cells and introducing a new assay for in vivo induction of apoptosis.
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Apoptosis , Neoplasias Pulmonares/fisiopatología , Neoplasias Pulmonares/secundario , Pulmón/irrigación sanguínea , Pulmón/patología , Melanoma/patología , Metástasis de la Neoplasia , Neovascularización Patológica , Neoplasias Cutáneas/patología , Animales , Supervivencia Celular , Fibroblastos , Humanos , Ratones , Células Neoplásicas Circulantes , Ratas , Factores de TiempoRESUMEN
We have reinvestigated the hypothesis of the relative importance of glomus cell plasma and mitochondrial membrane potentials (E(m) and psi(m), respectively) in acute hypoxia by a noninvasive fluorescence microimaging technique using the voltage-sensitive dyes bis-oxonol and JC-1, respectively. Short-term (24 h)-cultured rat glomus cells and cultured PC-12 cells were used for the study. Glomus cell E(m) depolarization was indirectly confirmed by an increase in bis-oxonol (an anionic probe) fluorescence due to a graded increase in extracellular K(+). Fluorescence responses of glomus cell E(m) to acute hypoxia (approximately 10 Torr Po(2)) indicated depolarization in 20%, no response in 45%, and hyperpolarization in 35% of the cells tested, whereas all PC-12 cells consistently depolarized in response to hypoxia. Furthermore, glomus cell E(m) hyperpolarization was confirmed with high CO (approximately 500 Torr). Glomus cell psi(m) depolarization was indirectly assessed by a decrease in JC-1 (a cationic probe) fluorescence. Accordingly, 1 microM carbonyl cyanide p-trifluoromethoxyphenylhydrazone (an uncoupler of oxidative phosphorylation), high CO (a metabolic inhibitor), and acute hypoxia (approximately 10 Torr Po(2)) consistently depolarized the mitochondria in all glomus cells tested. Likewise, all PC-12 cell mitochondria depolarized in response to FCCP and hypoxia. Thus, although bis-oxonol could not show glomus cell depolarization consistently, JC-1 monitored glomus cell mitochondrial depolarization as an inevitable phenomenon in hypoxia. Overall, these responses supported our "metabomembrane hypothesis" of chemoreception.
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Cuerpo Carotídeo/fisiología , Hipoxia/fisiopatología , Microscopía Fluorescente/métodos , Enfermedad Aguda , Animales , Bencimidazoles , Carbocianinas , Monóxido de Carbono/farmacología , Cuerpo Carotídeo/citología , Colorantes Fluorescentes , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Mitocondrias/fisiología , Oxígeno/farmacología , Células PC12 , Técnicas de Placa-Clamp , Potasio/farmacología , Ratas , Ratas Sprague-Dawley , TiobarbitúricosRESUMEN
These studies using both intact lung and reconstituted cell systems have shown that pulmonary endothelial cells respond rapidly (within several seconds) to the acute cessation of perfusate flow, i.e., ischemia. These effects represent a response to the loss of shear stress and are unrelated to changes in cellular oxygenation. The immediate response is partial depolarization of the endothelial cell membrane followed by activation of endothelial NADPH oxidase and the extracellular generation of superoxide anion. Dismutation of superoxide to H2O2 generates a cell signaling molecule that results in the activation of protein kinases and transcription factors which in turn lead to NO generation and activation of endothelial cell division. The presumed physiological role of this signal cascade is the generation of a vasodilator (NO) and the formation of new capillaries in the effort to restore blood flow.
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Endotelio Vascular/fisiopatología , Isquemia/fisiopatología , Pulmón/fisiopatología , Circulación Pulmonar/fisiología , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/fisiología , Animales , Modelos Animales de Enfermedad , Técnicas In Vitro , Pulmón/irrigación sanguínea , Ratones , RatasRESUMEN
Mitochondria can govern local concentrations of second messengers, such as reactive oxygen species (ROS), and mitochondrial translocation to discrete subcellular regions may contribute to this signaling function. Here, we report that exposure of pulmonary artery endothelial cells to hypoxia triggered a retrograde mitochondrial movement that required microtubules and the microtubule motor protein dynein and resulted in the perinuclear clustering of mitochondria. This subcellular redistribution of mitochondria was accompanied by the accumulation of ROS in the nucleus, which was attenuated by suppressing perinuclear clustering of mitochondria with nocodazole to destabilize microtubules or with small interfering RNA-mediated knockdown of dynein. Although suppression of perinuclear mitochondrial clustering did not affect the hypoxia-induced increase in the nuclear abundance of hypoxia-inducible factor 1α (HIF-1α) or the binding of HIF-1α to an oligonucleotide corresponding to a hypoxia response element (HRE), it eliminated oxidative modifications of the VEGF (vascular endothelial growth factor) promoter. Furthermore, suppression of perinuclear mitochondrial clustering reduced HIF-1α binding to the VEGF promoter and decreased VEGF mRNA accumulation. These findings support a model for hypoxia-induced transcriptional regulation in which perinuclear mitochondrial clustering results in ROS accumulation in the nucleus and causes oxidative base modifications in the VEGF HRE that are important for transcriptional complex assembly and VEGF mRNA expression.
Asunto(s)
Hipoxia de la Célula , Núcleo Celular/metabolismo , Mitocondrias/metabolismo , Oxidantes/metabolismo , Transcripción Genética , ADN/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , ARN Mensajero/genética , Especies Reactivas de Oxígeno/metabolismo , Factor A de Crecimiento Endotelial Vascular/genéticaAsunto(s)
Molécula de Adhesión Celular del Leucocito Activado/análisis , Cadherinas/análisis , Endotelio Vascular/química , Neoplasias Pulmonares/secundario , Proteínas de Neoplasias/análisis , Animales , Células Cultivadas , Endotelio Vascular/citología , Humanos , Neoplasias Pulmonares/irrigación sanguínea , Microcirculación , Análisis de Secuencia por Matrices de Oligonucleótidos , RatasAsunto(s)
Cuerpo Carotídeo/fisiopatología , Hipoxia/fisiopatología , Mitocondrias/fisiología , Animales , Cuerpo Carotídeo/patología , Membrana Celular/fisiología , Células Cultivadas , Colorantes Fluorescentes , Membranas Intracelulares/fisiología , Cinética , Potenciales de la Membrana/fisiología , Microscopía Fluorescente/métodos , Ratas , Transducción de Señal/fisiologíaRESUMEN
Vascularization of solid tumors is thought to occur by sprouting or intussusceptive angiogenesis, co-option of existing vessels, and vasculogenic mimicry after the onset of tumor hypoxia, when the tumor radius exceeds the oxygen diffusion distance. In contrast, here we show that individual endothelial cells that are incorporated into pre-hypoxic tumors give rise to tumor blood vessels via vasculogenesis. Small metastatic lung tumor sections obtained after tail-vein injection of a syngeneic breast cancer cell line in the nude mice were labeled with antibodies against endothelial cell markers. Immunofluorescence showed the incorporation and mixed growth of CD31-, Tie-2-, and CD105-positive endothelial cells in tumors with radii less than oxygen diffusion distance and subsequent development of blood vessels from these early-incorporated endothelial cells. This observation lays the foundation of a novel vasculogenic paradigm of tumor vascularization, where incorporation of endothelial cells and their growth among tumor cells occur before the onset of core hypoxia in lung metastatic tumors.