RESUMO
Pulmonary arterial hypertension (PAH) is a morbid disease characterized by significant lung endothelial cell (EC) dysfunction. Prior work has shown that microvascular endothelial cells (MVECs) isolated from animals with experimental PAH and patients with PAH exhibit significant abnormalities in metabolism and calcium signaling. With regards to metabolism, we and others have shown evidence of increased aerobic glycolysis and evidence of increased utilization of alternate fuel sources (such as fatty acids) in PAH EC. In the realm of calcium signaling, our prior work linked increased activity of the transient receptor potential vanilloid-4 (TRPV4) channel to increased proliferation of MVECs isolated from the Sugen/Hypoxia rat model of PAH (SuHx-MVECs). However, the relationship between metabolic shifts and calcium abnormalities was not clear. Specifically, whether shifts in metabolism were responsible for increasing TRPV4 channel activity in SuHx-MVECs was not known. In this study, using human data, serum samples from SuHx rats, and SuHx-MVECs, we describe the consequences of increased MVEC fatty acid oxidation in PAH. In human samples, we observed an increase in long-chain fatty acid levels that was associated with PAH severity. Next, using SuHx rats and SuHx-MVECs, we observed increased intracellular levels of lipids. We also show that increasing intracellular lipid content increases TRPV4 activity, whereas inhibiting fatty acid oxidation normalizes basal calcium levels in SuHx-MVECs. By exploring the fate of fatty acid-derived carbons, we observed that the metabolite linking increased intracellular lipids to TRPV4 activity was ß-hydroxybutyrate (BOHB), a product of fatty acid oxidation. Finally, we show that BOHB supplementation alone is sufficient to sensitize the TRPV4 channel in rat and mouse MVECs. Returning to humans, we observe a transpulmonary BOHB gradient in human patients with PAH. Thus, we establish a link between fatty acid oxidation, BOHB production, and TRPV4 activity in MVECs in PAH. These data provide new insight into metabolic regulation of calcium signaling in lung MVECs in PAH.NEW & NOTEWORTHY In this paper, we explore the link between metabolism and intracellular calcium levels in microvascular endothelial cells (MVECs) in pulmonary arterial hypertension (PAH). We show that fatty acid oxidation promotes sensitivity of the transient receptor potential vanilloid-4 (TRPV4) calcium channel in MVECs isolated from a rodent model of PAH.
Assuntos
Antineoplásicos , Hipertensão Arterial Pulmonar , Animais , Humanos , Camundongos , Ratos , Cálcio/metabolismo , Células Endoteliais/metabolismo , Hipertensão Pulmonar Primária Familiar/metabolismo , Ácidos Graxos/metabolismo , Lipídeos , Pulmão/metabolismo , Hipertensão Arterial Pulmonar/metabolismo , Canais de Cátion TRPV/metabolismoRESUMO
Pulmonary arterial hypertension (PAH) is a morbid disease characterized by progressive right ventricle (RV) failure due to elevated pulmonary artery pressures (PAP). In PAH, histologically complex vaso-occlusive lesions in the pulmonary vasculature contribute to elevated PAP. However, the mechanisms underlying dysfunction of the microvascular endothelial cells (MVECs) that comprise a significant portion of these lesions are not well understood. We recently showed that MVECs isolated from the Sugen/hypoxia (SuHx) rat experimental model of PAH (SuHx-MVECs) exhibit increases in migration/proliferation, mitochondrial reactive oxygen species (ROS; mtROS) production, intracellular calcium levels ([Ca2+]i), and mitochondrial fragmentation. Furthermore, quenching mtROS with the targeted antioxidant MitoQ attenuated basal [Ca2+]i, migration and proliferation; however, whether increased mtROS-induced [Ca2+]i entry affected mitochondrial morphology was not clear. In this study, we sought to better understand the relationship between increased ROS, [Ca2+]i, and mitochondrial morphology in SuHx-MVECs. We measured changes in mitochondrial morphology at baseline and following inhibition of mtROS, with the targeted antioxidant MitoQ, or transient receptor potential vanilloid-4 (TRPV4) channels, which we previously showed were responsible for mtROS-induced increases in [Ca2+]i in SuHx-MVECs. Quenching mtROS or inhibiting TRPV4 attenuated fragmentation in SuHx-MVECs. Conversely, inducing mtROS production in MVECs from normoxic rats (N-MVECs) increased fragmentation. Ca2+ entry induced by the TRPV4 agonist GSK1017920A was significantly increased in SuHx-MVECs and was attenuated with MitoQ treatment, indicating that mtROS contributes to increased TRPV4 activity in SuHx-MVECs. Basal and maximal respiration were depressed in SuHx-MVECs, and inhibiting mtROS, but not TRPV4, improved respiration in these cells. Collectively, our data show that, in SuHx-MVECs, mtROS production promotes TRPV4-mediated increases in [Ca2+]i, mitochondrial fission, and decreased mitochondrial respiration. These results suggest an important role for mtROS in driving MVEC dysfunction in PAH.
Assuntos
Células Endoteliais/patologia , Hipóxia/complicações , Indóis/toxicidade , Pulmão/patologia , Mitocôndrias/patologia , Hipertensão Arterial Pulmonar/patologia , Pirróis/toxicidade , Espécies Reativas de Oxigênio/metabolismo , Inibidores da Angiogênese/toxicidade , Animais , Cálcio/metabolismo , Células Cultivadas , Células Endoteliais/metabolismo , Pulmão/metabolismo , Masculino , Mitocôndrias/metabolismo , Consumo de Oxigênio , Hipertensão Arterial Pulmonar/etiologia , Hipertensão Arterial Pulmonar/metabolismo , Ratos , Ratos Wistar , Remodelação VascularRESUMO
Noncanonical roles for caspase-3 are emerging in the fields of cancer and developmental biology. However, little is known of nonapoptotic functions of caspase-3 in most cell types. We have recently demonstrated a disassociation between caspase-3 activation and execution of apoptosis with accompanying cytoplasmic caspase-3 sequestration and preserved endothelial barrier function. Therefore, we tested the hypothesis that nonapoptotic caspase-3 activation promotes endothelial barrier integrity. Human lung microvascular endothelial cells were exposed to thrombin, a nonapoptotic stimulus, and endothelial barrier function was assessed using electric cell-substrate impedance sensing. Actin cytoskeletal rearrangement and paracellular gap formation were assessed using phalloidin staining. Cell stiffness was evaluated using magnetic twisting cytometry. In addition, cell lysates were harvested for protein analyses. Caspase-3 was inhibited pharmacologically with pan-caspase and a caspase-3-specific inhibitor. Molecular inhibition of caspase-3 was achieved using RNA interference. Cells exposed to thrombin exhibited a cytoplasmic activation of caspase-3 with transient and nonapoptotic decrease in endothelial barrier function as measured by a drop in electrical resistance followed by a rapid recovery. Inhibition of caspases led to a more pronounced and rapid drop in thrombin-induced endothelial barrier function, accompanied by increased endothelial cell stiffness and paracellular gaps. Caspase-3-specific inhibition and caspase-3 knockdown both resulted in more pronounced thrombin-induced endothelial barrier disruption. Taken together, our results suggest cytoplasmic caspase-3 has nonapoptotic functions in human endothelium and can promote endothelial barrier integrity.
Assuntos
Caspase 3/metabolismo , Células Endoteliais/citologia , Endotélio Vascular/metabolismo , Mucosa Respiratória/citologia , Junções Íntimas/efeitos dos fármacos , Citoesqueleto de Actina/fisiologia , Permeabilidade Capilar/efeitos dos fármacos , Caspase 3/genética , Células Cultivadas , Impedância Elétrica , Endotélio Vascular/citologia , Humanos , Pulmão/citologia , Interferência de RNA , RNA Interferente Pequeno/genética , Trombina/farmacologiaRESUMO
Pulmonary arterial hypertension (PAH) is a lethal disease characterized by elevations in pulmonary arterial pressure, in part due to formation of occlusive lesions in the distal arterioles of the lung. These complex lesions may comprise multiple cell types, including endothelial cells (ECs). To better understand the molecular mechanisms underlying EC dysfunction in PAH, lung microvascular endothelial cells (MVECs) were isolated from normoxic rats (N-MVECs) and rats subjected to SU5416 plus hypoxia (SuHx), an experimental model of PAH. Compared with N-MVECs, MVECs isolated from SuHx rats (SuHx-MVECs) appeared larger and more spindle shaped morphologically and expressed canonical smooth muscle cell markers smooth muscle-specific α-actin and myosin heavy chain in addition to endothelial markers such as Griffonia simplicifolia and von Willebrand factor. SuHx-MVEC mitochondria were dysfunctional, as evidenced by increased fragmentation/fission, decreased oxidative phosphorylation, and increased reactive oxygen species (ROS) production. Functionally, SuHx-MVECs exhibited increased basal levels of intracellular calcium concentration ([Ca2+]i) and enhanced migratory and proliferative capacity. Treatment with global (TEMPOL) or mitochondria-specific (MitoQ) antioxidants decreased ROS levels and basal [Ca2]i in SuHx-MVECs. TEMPOL and MitoQ also decreased migration and proliferation in SuHx-MVECs. Additionally, inhibition of ROS-induced Ca2+ entry via pharmacologic blockade of transient receptor potential vanilloid-4 (TRPV4) attenuated [Ca2]i, migration, and proliferation. These findings suggest a role for mitochondrial ROS-induced Ca2+ influx via TRPV4 in promoting abnormal migration and proliferation in MVECs in this PAH model.
Assuntos
Cálcio/metabolismo , Endotélio Vascular/patologia , Hipertensão Pulmonar/patologia , Hipóxia/fisiopatologia , Indóis/toxicidade , Artéria Pulmonar/patologia , Pirróis/toxicidade , Canais de Cátion TRPV/metabolismo , Doenças Vasculares/patologia , Inibidores da Angiogênese/toxicidade , Animais , Células Cultivadas , Endotélio Vascular/metabolismo , Hipertensão Pulmonar/etiologia , Hipertensão Pulmonar/metabolismo , Masculino , Microvasos/metabolismo , Microvasos/patologia , Artéria Pulmonar/metabolismo , Ratos , Ratos Wistar , Canais de Cátion TRPV/genética , Doenças Vasculares/etiologia , Doenças Vasculares/metabolismoRESUMO
Elevated levels of reactive oxygen species and intracellular Ca2+ play a key role in endothelial barrier dysfunction in acute lung injury. We previously showed that H2O2-induced increases in intracellular calcium concentrations ([Ca2+]i) in lung microvascular endothelial cells (LMVECs) involve the membrane Ca2+ channel, transient receptor potential vanilloid-4 (TRPV4) and that inhibiting this channel attenuated H2O2-induced barrier disruption in vitro. We also showed that phosphorylation of TRPV4 by the Src family kinase, Fyn, contributes to H2O2-induced Ca2+ influx in LMVEC. In endothelial cells, Fyn is tethered to the cell membrane by CD36, a fatty acid transporter. In this study, we assessed the effect of genetic loss or pharmacological inhibition of CD36 on Ca2+ responses to H2O2 H2O2-induced Ca2+ influx was attenuated in LMVEC isolated from mice lacking CD36 (CD36-/-). TRPV4 expression and function was unchanged in LMVEC isolated from wild-type (WT) and CD36-/- mice, as well as mice with deficiency for Fyn (Fyn-/-). TRPV4 immunoprecipitated with Fyn, but this interaction was decreased in CD36-/- LMVEC. The amount of phosphorylated TRPV4 was decreased in LMVEC from CD36-/- mice compared with WT controls. Loss of CD36 altered subcellular localization of Fyn, while inhibition of CD36 fatty acid transport with succinimidyl oleate did not attenuate H2O2-induced Ca2+ influx. Lastly, we found that CD36-/- mice were protected from ischemia-reperfusion injury in vivo. In conclusion, our data suggest that CD36 plays an important role in H2O2-mediated lung injury and that the mechanism may involve CD36-dependent scaffolding of Fyn to the cell membrane to facilitate TRPV4 phosphorylation.
Assuntos
Antígenos CD36/metabolismo , Cálcio/metabolismo , Células Endoteliais/metabolismo , Peróxido de Hidrogênio/farmacologia , Pulmão/irrigação sanguínea , Microvasos/citologia , Animais , Células Endoteliais/efeitos dos fármacos , Ácidos Graxos/metabolismo , Deleção de Genes , Lipoproteínas LDL/farmacologia , Camundongos Endogâmicos C57BL , Ácido Oleico/farmacologia , Fosforilação/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-fyn/metabolismo , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/patologia , Frações Subcelulares/efeitos dos fármacos , Frações Subcelulares/metabolismo , Canais de Cátion TRPV/metabolismoRESUMO
In acute respiratory distress syndrome, both reactive oxygen species (ROS) and increased intracellular calcium ([Ca(2+)]i) are thought to play important roles in promoting endothelial paracellular permeability, but the mechanisms linking ROS and [Ca(2+)]i in microvascular endothelial cells are not known. In this study, we assessed the effect of hydrogen peroxide (H2O2) on [Ca(2+)]i in mouse and human lung microvascular endothelial cells (MLMVEC and HLMVEC, respectively). We found that in both MLMVECs and HLMVECs, exogenously applied H2O2 increased [Ca(2+)]i through Ca(2+) influx and that pharmacologic inhibition of the calcium channel transient receptor potential vanilloid 4 (TRPV4) attenuated the H2O2-induced Ca(2+) influx. Additionally, knockdown of TRPV4 in HLMVEC also attenuated calcium influx following H2O2 challenge. Administration of H2O2 or TRPV4 agonists decreased transmembrane electrical resistance (TER), suggesting increased barrier permeability. To explore the regulatory mechanisms underlying TRPV4 activation by ROS, we examined H2O2-induced Ca(2+) influx in MLMVECs and HLMVECs with either genetic deletion, silencing, or pharmacologic inhibition of Fyn, a Src family kinase. In both MLMVECs derived from mice deficient for Fyn and HLMVECs treated with either siRNA targeted to Fyn or the Src family kinase inhibitor SU-6656 for 24 or 48 h, the H2O2-induced Ca(2+) influx was attenuated. Treatment with SU-6656 decreased the levels of phosphorylated, but not total, TRPV4 protein and had no effect on TRPV4 response to the external agonist, GSK1016790A. In conclusion, our data suggest that application of exogenous H2O2 increases [Ca(2+)]i and decreases TER in microvascular endothelial cells via activation of TRPV4 through a mechanism that requires the Src kinase Fyn.
Assuntos
Cálcio/metabolismo , Células Endoteliais/metabolismo , Endotélio Vascular/metabolismo , Peróxido de Hidrogênio/farmacologia , Pulmão/metabolismo , Microvasos/metabolismo , Canais de Cátion TRPV/metabolismo , Animais , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Células Cultivadas , Células Endoteliais/efeitos dos fármacos , Endotélio Vascular/efeitos dos fármacos , Humanos , Leucina/análogos & derivados , Leucina/farmacologia , Pulmão/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microvasos/efeitos dos fármacos , Permeabilidade/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-fyn/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Sulfonamidas/farmacologia , Quinases da Família src/metabolismoRESUMO
Oxidant injury contributes to acute lung injury (ALI). We previously reported that activation of protein kinase GI (PKGI) posttranscriptionally increased the key antioxidant enzymes catalase and glutathione peroxidase 1 (Gpx-1) and attenuated oxidant-induced cytotoxicity in mouse lung microvascular endothelial cells (MLMVEC). The present studies tested the hypothesis that the antioxidant effect of PKGI is mediated via inhibition of the c-Abl tyrosine kinase. We found that activation of PKGI with the cGMP analog 8pCPT-cGMP inhibited c-Abl activity and decreased c-Abl expression in wild-type but not PKGI(-/-) MLMVEC. Treatment of wild-type MLMVEC with atrial natriuretic peptide also inhibited c-Abl activation. Moreover, treatment of MLMVEC with the c-Abl inhibitor imatinib increased catalase and GPx-1 protein in a posttranscriptional fashion. In imatinib-treated MLMVEC, there was no additional effect of 8pCPT-cGMP on catalase or GPx-1. The imatinib-induced increase in antioxidant proteins was associated with an increase in extracellular H2O2 scavenging by MLMVEC, attenuation of oxidant-induced endothelial barrier dysfunction, and prevention of oxidant-induced endothelial cell death. Finally, in the isolated perfused lung, imatinib prevented oxidant-induced endothelial toxicity. We conclude that cGMP, through activation of PKGI, inhibits c-Abl, leading to increased key antioxidant enzymes and resistance to lung endothelial oxidant injury. Inhibition of c-Abl by active PKGI may be the downstream mechanism underlying PKGI-mediated antioxidant signaling. Tyrosine kinase inhibitors may represent a novel therapeutic approach in oxidant-induced ALI.
Assuntos
Lesão Pulmonar Aguda/metabolismo , Proteínas Quinases Dependentes de GMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Pulmão/metabolismo , Proteínas Proto-Oncogênicas c-abl/antagonistas & inibidores , Transporte Ativo do Núcleo Celular/fisiologia , Animais , Apoptose/efeitos dos fármacos , Fator Natriurético Atrial/metabolismo , Benzamidas/farmacologia , Catalase/metabolismo , Células Cultivadas , GMP Cíclico/análogos & derivados , GMP Cíclico/farmacologia , Proteínas Quinases Dependentes de GMP Cíclico/efeitos dos fármacos , Proteínas Quinases Dependentes de GMP Cíclico/genética , Células Endoteliais/metabolismo , Ativação Enzimática , Glutationa Peroxidase/metabolismo , Peróxido de Hidrogênio/metabolismo , Mesilato de Imatinib , Pulmão/citologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Oxirredução/efeitos dos fármacos , Piperazinas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-abl/metabolismo , Pirimidinas/farmacologia , RNA Mensageiro/biossíntese , Transdução de Sinais/efeitos dos fármacos , Glutationa Peroxidase GPX1RESUMO
Electrical cell-substrate impedance sensing (ECIS) is an in vitro methodology for measuring the barrier integrity of a variety of cell types, including pulmonary endothelial cells. These experiments are frequently used for in vitro assessment of lung injury. The data derived from ECIS experiments consists of repeated measures of resistance across an endothelial monolayer. As such, these data reflect the dynamic changes in electrical resistance that occur over time. Currently methodologies for assessing ECIS data rely on single point assessments of barrier function, such as the maximal drop in trans-endothelial electrical resistance (TERMax ). However, this approach ignores the myriad of changes in resistance that occur before and after the TERMax data point. Herein, we utilize polynomial curve fitting on experimentally generated ECIS data, thus allowing for comparing ECIS experiments by examining the mean polynomial coefficients between groups. We show that polynomial curves accurately fit a variety of ECIS data, and that concordance between TERMax and coefficient analysis varies by type of stimulus, suggesting that TERMax differences may not always correlate with a significant difference in the overall shape of the ECIS profile. Lastly, we identify factors that impact coefficient values obtained in our analyses, including the length of time devoted to baseline measurements before addition of stimuli. Polynomial coefficient analysis is another tool that can be used for more comprehensive interrogation of ECIS data to better understand the biological underpinnings that lead to changes in barrier dysfunction in vitro.
Assuntos
Membrana Celular/fisiologia , Células Endoteliais/citologia , Pulmão/citologia , Animais , Técnicas Biossensoriais , Impedância Elétrica , Células Endoteliais/fisiologia , Humanos , Pulmão/fisiologia , CamundongosRESUMO
Increasing evidence suggests that endothelial cytotoxicity from reactive oxygen species (ROS) contributes to the pathogenesis of acute lung injury. Treatments designed to increase intracellular cGMP attenuate ROS-mediated apoptosis and necrosis in several cell types, but the mechanisms are not understood, and the effect of cGMP on pulmonary endothelial cell death remains controversial. In the current study, increasing intracellular cGMP by either 8pCPT-cGMP (50 microM) or atrial natriuretic peptide (10 nM) significantly attenuated cell death in H(2)O(2)-challenged mouse lung microvascular (MLMVEC) monolayers. 8pCPT-cGMP also decreased perfusate LDH release in isolated mouse lungs exposed to H(2)O(2) or ischemia-reperfusion. The protective effect of increasing cGMP in MLMVECs was accompanied by enhanced endothelial H(2)O(2) scavenging (measured by H(2)O(2) electrode) and decreased intracellular ROS concentration (measured by 2',7'-dichlorofluorescin fluorescence) as well as decreased phosphorylation of p38 MAPK and Akt. The cGMP-mediated cytoprotection and increased H(2)O(2) scavenging required >2 h of 8pCPT-cGMP incubation in wild-type MLMVEC and were absent in MLMVEC from protein kinase G (PKG(I))-/- mice suggesting a PKG(I)-mediated effect on gene regulation. Catalase and glutathione peroxidase 1 (Gpx-1) protein were increased by cGMP in wild-type but not PKG(I)-/- MLMVEC monolayers. Both the cGMP-mediated increases in antioxidant proteins and H(2)O(2) scavenging were prevented by inhibition of translation with cycloheximide. 8pCPT-cGMP had minimal effects on catalase and Gpx-1 mRNA. We conclude that cGMP, through PKG(I), attenuated H(2)O(2)-induced cytotoxicity in MLMVEC by increasing catalase and Gpx-1 expression through an unknown posttranscriptional effect.
Assuntos
Antioxidantes/metabolismo , Proteínas Quinases Dependentes de GMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Células Endoteliais/metabolismo , Peróxido de Hidrogênio/farmacologia , Pulmão/irrigação sanguínea , Microvasos/fisiopatologia , Oxidantes/farmacologia , Animais , Fator Natriurético Atrial/farmacologia , Catalase/metabolismo , Morte Celular , Células Cultivadas , GMP Cíclico/análogos & derivados , GMP Cíclico/farmacologia , Proteína Quinase Dependente de GMP Cíclico Tipo I , Proteínas Quinases Dependentes de GMP Cíclico/deficiência , Células Endoteliais/efeitos dos fármacos , Glutationa Peroxidase/metabolismo , Membranas Intracelulares/metabolismo , Isoenzimas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Oxirredutases/metabolismo , Processamento de Proteína Pós-Traducional , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Tionucleotídeos/farmacologiaRESUMO
Ischemia-reperfusion (I/R) lung injury is characterized by increased pulmonary endothelial permeability and edema, but the genetic basis for this injury is unknown. We utilized an in vivo mouse preparation of unilateral lung I/R to evaluate the genetic determinants of I/R lung injury. An index of pulmonary vascular protein permeability was measured by the ratio of left-to-right lung Evans blue dye of eight inbred mouse strains after 30 min of left lung ischemia and 150 min of reperfusion. The order of strain-specific sensitivity to I/R lung injury was BALB/c < SJL/J < CBA/J < C57BL/6J < 129/J < A/J < C3H/H3J < SWR/J. The reciprocal F1 offspring of the BALB/c and SWR/J progenitor strains had intermediate phenotypes but a differing variance. A similar pattern of right lung Evans blue dye content suggested the presence of contralateral injury because baseline vascular permeability was not different. Lung I/R injury was attenuated by NADPH oxidase inhibition, indicating a role for NADPH oxidase-derived reactive oxygen species (ROS). There was no strain-dependent difference in lung NADPH oxidase expression. Strain-related differences in zymosan-stimulated neutrophil ROS production did not correlate with I/R lung injury in that neutrophil ROS production in SWR/J mice was greater than C57BL/6J but not different from BALB/c mice. These data indicate the presence of a genetic sensitivity to lung I/R injury that involves multiple genes including a maternal-related factor. Although neutrophil-derived ROS production is also modulated by genetic factors, the pattern did not explain the genetic sensitivity to lung I/R injury.
Assuntos
Predisposição Genética para Doença , Traumatismo por Reperfusão/genética , Traumatismo por Reperfusão/fisiopatologia , Animais , Permeabilidade Capilar/fisiologia , Regulação Enzimológica da Expressão Gênica , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Camundongos Endogâmicos , NADPH Oxidases/genética , NADPH Oxidases/fisiologia , Neutrófilos/enzimologia , Espécies Reativas de Oxigênio/metabolismo , Especificidade da Espécie , Fatores de Tempo , Zimosan/farmacologiaRESUMO
Severe sepsis and septic shock are frequent causes of the acute respiratory distress syndrome, and important sources of human mortality. Lipopolysaccharide (LPS), a component of Gram-negative bacterial cell walls, plays a major role in the pathogenesis of severe sepsis and septic shock. LPS exposure induces the production of harmful reactive oxygen species, and the resultant oxidant injury has been implicated in the pathogenesis of both severe sepsis and ARDS. We previously showed that the tyrosine kinase inhibitor imatinib increases lung endothelial antioxidant enzymes and protects against pulmonary endothelial antioxidant injury. In the present study, we tested the hypothesis that imatinib would protect against lung injury and systemic inflammation caused by intravenous LPS in an intact mouse model of endotoxemia mimicking early sepsis. We found that intravenous LPS induced a significant increase in the activity of lung xanthine oxidoreductase (XOR), an enzyme which is a major source of reactive oxygen species and implicated in the pathogenesis of acute lung injury. Imatinib had no effect of LPS-induced XOR activity. However, pretreatment of mice with imatinib increased lung catalase activity and decreased intravenous LPS-induced lung oxidant injury as measured by γ-H2AX, a marker of oxidant-induced DNA damage, lung apoptosis, and pulmonary edema. Imatinib also attenuated systemic cytokine expression after intravenous LPS exposure. Finally, imatinib completely prevented mortality in an in vivo, intravenous LPS mouse model of endotoxemia and lung injury. These results support the testing of imatinib as a novel pharmacologic agent in the treatment of Gram-negative sepsis and sepsis-induced ARDS.
RESUMO
Severe malaria can trigger acute lung injury characterized by pulmonary edema resulting from increased endothelial permeability. However, the mechanism through which lung fluid conductance is altered during malaria remains unclear. To define the role that the scavenger receptor CD36 may play in mediating this response, C57BL/6J (WT) and CD36-/- mice were infected with P. berghei ANKA and monitored for changes in pulmonary endothelial barrier function employing an isolated perfused lung system. WT lungs demonstrated a >10-fold increase in two measures of paracellular fluid conductance and a decrease in the albumin reflection coefficient (σalb) compared to control lungs indicating a loss of barrier function. In contrast, malaria-infected CD36-/- mice had near normal fluid conductance but a similar reduction in σalb. In WT mice, lung sequestered iRBCs demonstrated production of reactive oxygen species (ROS). To determine whether knockout of CD36 could protect against ROS-induced endothelial barrier dysfunction, mouse lung microvascular endothelial monolayers (MLMVEC) from WT and CD36-/- mice were exposed to H2O2. Unlike WT monolayers, which showed dose-dependent decreases in transendothelial electrical resistance (TER) from H2O2 indicating loss of barrier function, CD36-/- MLMVEC demonstrated dose-dependent increases in TER. The differences between responses in WT and CD36-/- endothelial cells correlated with important differences in the intracellular compartmentalization of the CD36-associated Fyn kinase. Malaria infection increased total lung Fyn levels in CD36-/- lungs compared to WT, but this increase was due to elevated production of the inactive form of Fyn further suggesting a dysregulation of Fyn-mediated signaling. The importance of Fyn in CD36-dependent endothelial signaling was confirmed using in vitro Fyn knockdown as well as Fyn-/- mice, which were also protected from H2O2- and malaria-induced lung endothelial leak, respectively. Our results demonstrate that CD36 and Fyn kinase are critical mediators of the increased lung endothelial fluid conductance caused by malaria infection.
Assuntos
Células Epiteliais Alveolares/metabolismo , Barreira Alveolocapilar/metabolismo , Antígenos CD36/metabolismo , Malária/metabolismo , Plasmodium berghei , Proteínas Proto-Oncogênicas c-fyn/metabolismo , Células Epiteliais Alveolares/patologia , Animais , Barreira Alveolocapilar/fisiopatologia , Antígenos CD36/genética , Eritrócitos/metabolismo , Eritrócitos/parasitologia , Técnicas de Silenciamento de Genes , Pulmão/metabolismo , Pulmão/patologia , Malária/patologia , Camundongos , Camundongos Knockout , Permeabilidade , Espécies Reativas de Oxigênio/metabolismo , Transdução de SinaisRESUMO
Increased pulmonary endothelial cGMP was shown to prevent endothelial barrier dysfunction through activation of protein kinase G (PKG(I)). Vasodilator-stimulated phosphoprotein (VASP) has been hypothesized to mediate PKG(I) barrier protection because VASP is a cytoskeletal phosphorylation target of PKG(I) expressed in cell-cell junctions. Unphosphorylated VASP was proposed to increase paracellular permeability through actin polymerization and stress fiber bundling, a process inhibited by PKG(I)-mediated phosphorylation of Ser(157) and Ser(239). To test this hypothesis, we examined the role of VASP in the transient barrier dysfunction caused by H(2)O(2) in human pulmonary artery endothelial cell (HPAEC) monolayers studied without and with PKG(I) expression introduced by adenoviral infection (Ad.PKG). In the absence of PKG(I) expression, H(2)O(2) (100-250 microM) caused a transient increased permeability and pSer(157)-VASP formation that were both attenuated by protein kinase C inhibition. Potentiation of VASP Ser(157) phosphorylation by either phosphatase 2B inhibition with cyclosporin or protein kinase A activation with forskolin prolonged, rather than inhibited, the increased permeability caused by H(2)O(2). With Ad.PKG infection, inhibition of VASP expression with small interfering RNA exacerbated H(2)O(2)-induced barrier dysfunction but had no effect on cGMP-mediated barrier protection. In addition, expression of a Ser-double phosphomimetic mutant VASP failed to reproduce the protective effects of activated PKG(I). Finally, expression of a Ser-double phosphorylation-resistant VASP failed to interfere with the ability of cGMP/PKG(I) to attenuate H(2)O(2)-induced disruption of VE-cadherin homotypic binding. Our results suggest that VASP phosphorylation does not explain the protective effect of cGMP/PKG(I) on H(2)O(2)-induced endothelial barrier dysfunction in HPAEC.
Assuntos
GMP Cíclico/farmacologia , Endotélio Vascular/fisiologia , Fosfoproteínas/metabolismo , Artéria Pulmonar/fisiologia , Vasodilatadores/farmacologia , Colforsina/farmacologia , Ciclosporina/farmacologia , Endotélio Vascular/citologia , Endotélio Vascular/efeitos dos fármacos , Humanos , Peróxido de Hidrogênio/farmacologia , Cinética , Fosfosserina/metabolismo , Plasmídeos , Artéria Pulmonar/citologia , Artéria Pulmonar/efeitos dos fármacos , RNA Interferente Pequeno/genética , TransfecçãoRESUMO
High tidal volume (HV(T)) ventilation causes pulmonary endothelial barrier dysfunction. HV(T) ventilation also increases lung nitric oxide (NO) and cGMP. NO contributes to HV(T) lung injury, but the role of cGMP is unknown. In the current study, ventilation of isolated C57BL/6 mouse lungs increased perfusate cGMP as a function of V(T). Ventilation with 20 ml/kg V(T) for 80 min increased the filtration coefficient (K(f)), an index of vascular permeability. The increased cGMP and K(f) caused by HV(T) were attenuated by nitric oxide synthase (NOS) inhibition and in lungs from endothelial NOS knockout mice. Inhibition of soluble guanylyl cyclase (sGC) in wild-type lungs (10 muM ODQ) also blocked cGMP generation and inhibited the increase in K(f), suggesting an injurious role for sGC-derived cGMP. sGC inhibition also attenuated lung Evans blue dye albumin extravasation and wet-to-dry weight ratio in an anesthetized mouse model of HV(T) injury. Additional activation of sGC (1.5 muM BAY 41-2272) in isolated lungs at 40 min increased cGMP production and K(f) in lungs ventilated with 15 ml/kg V(T). HV(T) endothelial barrier dysfunction was attenuated with a nonspecific phosphodiesterase (PDE) inhibitor (100 muM IBMX) as well as an inhibitor (10 muM BAY 60-7550) specific for the cGMP-stimulated PDE2A. Concordantly, we found a V(T)-dependent increase in lung cAMP hydrolytic activity and PDE2A protein expression with a decrease in lung cAMP concentration that was blocked by BAY 60-7550. We conclude that HV(T)-induced endothelial barrier dysfunction resulted from a simultaneous increase in NO/sGC-derived cGMP and PDE2A expression causing decreased cAMP.
Assuntos
Nucleotídeo Cíclico Fosfodiesterase do Tipo 2/biossíntese , Guanilato Ciclase/metabolismo , Lesão Pulmonar Induzida por Ventilação Mecânica/enzimologia , 1-Metil-3-Isobutilxantina/farmacologia , Animais , Barreira Alveolocapilar , AMP Cíclico/genética , AMP Cíclico/metabolismo , GMP Cíclico/genética , GMP Cíclico/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 2/genética , Modelos Animais de Doenças , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/genética , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/genética , Guanilato Ciclase/genética , Camundongos , Camundongos Knockout , Óxido Nítrico/genética , Óxido Nítrico/metabolismo , Inibidores de Fosfodiesterase/farmacologia , Lesão Pulmonar Induzida por Ventilação Mecânica/genéticaRESUMO
Ischemia-reperfusion (IR) causes human lung injury in association with the release of atrial and brain natriuretic peptides (ANP and BNP), but the role of ANP/BNP in IR lung injury is unknown. ANP and BNP bind to natriuretic peptide receptor-A (NPR-A) generating cGMP and to NPR-C, a clearance receptor that can decrease intracellular cAMP. To determine the role of NPR-A signaling in IR lung injury, we administered the NPR-A blocker anantin in an in vivo SWR mouse preparation of unilateral lung IR. With uninterrupted ventilation, the left pulmonary artery was occluded for 30 min and then reperfused for 60 or 150 min. Anantin administration decreased IR-induced Evans blue dye extravasation and wet weight in the reperfused left lung, suggesting an injurious role for NPR-A signaling in lung IR. In isolated mouse lungs, exogenous ANP (2.5 nM) added to the perfusate significantly increased the filtration coefficient sevenfold only if lungs were subjected to IR. This effect of ANP was also blocked by anantin. Unilateral in vivo IR increased endogenous plasma ANP, lung cGMP concentration, and lung protein kinase G (PKG(I)) activation. Anantin enhanced plasma ANP concentrations and attenuated the increase in cGMP and PKG(I) activation but had no effect on lung cAMP. These data suggest that lung IR triggered ANP release and altered endothelial signaling so that NPR-A activation caused increased pulmonary endothelial permeability.
Assuntos
Guanilato Ciclase/fisiologia , Receptores do Fator Natriurético Atrial/fisiologia , Traumatismo por Reperfusão/fisiopatologia , Síndrome do Desconforto Respiratório/fisiopatologia , Animais , Fator Natriurético Atrial/metabolismo , Fator Natriurético Atrial/farmacologia , Moléculas de Adesão Celular/efeitos dos fármacos , Moléculas de Adesão Celular/fisiologia , AMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Pulmão/efeitos dos fármacos , Pulmão/fisiologia , Pulmão/fisiopatologia , Masculino , Camundongos , Camundongos Endogâmicos , Proteínas dos Microfilamentos/efeitos dos fármacos , Proteínas dos Microfilamentos/fisiologia , Perfusão , Fosfoproteínas/efeitos dos fármacos , Fosfoproteínas/fisiologia , Transdução de SinaisRESUMO
Increases in endothelial cGMP prevent oxidant-mediated endothelial barrier dysfunction, but the downstream mechanisms remain unclear. To determine the role of cGMP-dependent protein kinase (PKG)(I), human pulmonary artery endothelial cells (HPAEC) lacking PKG(I) expression were infected with a recombinant adenovirus encoding PKG(Ibeta) (Ad.PKG) and compared with uninfected and control-infected (Ad.betagal) HPAEC. Transendothelial electrical resistance (TER), an index of permeability, was measured after H(2)O(2) (250 microM) exposure with or without pretreatment with 8-(4-chlorophenylthio)guanosine 3',5'-cyclic monophosphate (CPT-cGMP). HPAEC infected with Ad.PKG, but not Ad.betagal, expressed PKG(I) protein and demonstrated Ser(239) and Ser(157) phosphorylation of vasodilator-stimulated phosphoprotein after treatment with CPT-cGMP. Adenoviral infection decreased basal permeability equally in Ad.PKG- and Ad.betagal-infected HPAEC compared with uninfected cells. Treatment with CPT-cGMP (100 microM) caused a PKG(I)-independent decrease in permeability (8.2 +/- 0.6%). In all three groups, H(2)O(2) (250 microM) caused a similar approximately 35% increase in permeability associated with increased actin stress fiber formation, intercellular gaps, loss of membrane VE-cadherin, and increased intracellular Ca(2+) concentration ([Ca(2+)](i)). In uninfected and Ad.betagal-infected HPAEC, pretreatment with CPT-cGMP (100 microM) partially blocked the increased permeability induced by H(2)O(2). In Ad.PKG-infected HPAEC, CPT-cGMP (50 microM) prevented the H(2)O(2)-induced TER decrease, cytoskeletal rearrangement, and loss of junctional VE-cadherin. CPT-cGMP attenuated the peak [Ca(2+)](i) caused by H(2)O(2) similarly (23%) in Ad.betagal- and Ad.PKG-infected HPAEC, indicating a PKG(I)-independent effect. These data suggest that cGMP decreased HPAEC basal permeability by a PKG(I)-independent process, whereas the ability of cGMP to prevent H(2)O(2)-induced barrier dysfunction was predominantly mediated by PKG(I) through a Ca(2+)-independent mechanism.