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1.
ERJ Open Res ; 10(1)2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-38410714

RESUMO

Background: Sepsis is a life-threatening condition that results from a dysregulated host response to infection, leading to organ dysfunction. Despite the prevalence and associated socioeconomic costs, treatment of sepsis remains limited to antibiotics and supportive care, and a majority of intensive care unit (ICU) survivors develop long-term cognitive complications post-discharge. The present study identifies a novel regulatory relationship between amyloid-ß (Aß) and the inflammasome-caspase-1 axis as key innate immune mediators that define sepsis outcomes. Methods: Medical ICU patients and healthy individuals were consented for blood and clinical data collection. Plasma cytokine, caspase-1 and Aß levels were measured. Data were compared against indices of multiorgan injury and other clinical parameters. Additionally, recombinant proteins were tested in vitro to examine the effect of caspase-1 on a functional hallmark of Aß, namely aggregation. Results: Plasma caspase-1 levels displayed the best predictive value in discriminating ICU patients with sepsis from non-infected ICU patients (area under the receiver operating characteristic curve=0.7080). Plasma caspase-1 and the Aß isoform Aßx-40 showed a significant positive correlation and Aßx-40 associated with organ injury. Additionally, Aß plasma levels continued to rise from time of ICU admission to 7 days post-admission. In silico, Aß harbours a predicted caspase-1 cleavage site, and in vitro studies demonstrated that caspase-1 cleaved Aß to inhibit its auto-aggregation, suggesting a novel regulatory relationship. Conclusions: Aßx-40 and caspase-1 are potentially useful early indicators of sepsis and its attendant organ injury. Additionally, Aßx-40 has emerged as a potential culprit in the ensuing development of post-ICU syndrome.

2.
MicroPubl Biol ; 20232023.
Artigo em Inglês | MEDLINE | ID: mdl-37662051

RESUMO

SARS-CoV-2 infection can result in a range of outcomes from asymptomatic/mild disease to severe COVID-19/fatality. In this study, we investigated the differential expression of small noncoding RNAs (sncRNAs) between patient cohorts defined by disease severity. We collected plasma samples, stratified these based on clinical outcomes, and sequenced their circulating sncRNAs. Excitingly, we found YRNA HY4 displays significant differential expression (p=0.025) between patients experiencing mild and severe disease. In agreement with recent reports identifying plasma YRNAs as indicators of influenza infection severity, our results strongly suggest that circulating HY4 levels represent a powerful prognostic indicator of likely SARS-CoV-2 patient infection outcome.

3.
Am J Respir Cell Mol Biol ; 65(6): 630-645, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34251286

RESUMO

Low tidal volume ventilation protects the lung in mechanically ventilated patients. The impact of the accompanying permissive hypoxemia and hypercapnia on endothelial cell recovery from injury is poorly understood. CA (carbonic anhydrase) IX is expressed in pulmonary microvascular endothelial cells (PMVECs), where it contributes to CO2 and pH homeostasis, bioenergetics, and angiogenesis. We hypothesized that CA IX is important for PMVEC survival and that CA IX expression and release from PMVECs are increased during infection. Although the plasma concentration of CA IX was unchanged in human and rat pneumonia, there was a trend toward increasing CA IX in the bronchoalveolar fluid of mechanically ventilated critically ill patients with pneumonia and a significant increase in CA IX in the lung tissue lysates of pneumonia rats. To investigate the functional implications of the lung CA IX increase, we generated PMVEC cell lines harboring domain-specific CA IX mutations. By using these cells, we found that infection promotes intracellular (IC) expression, release, and MMP (metalloproteinase)-mediated extracellular cleavage of CA IX in PMVECs. IC domain deletion uniquely impaired CA IX membrane localization. Loss of the CA IX IC domain promoted cell death after infection, suggesting that the IC domain has an important role in PMVEC survival. We also found that hypoxia improves survival, whereas hypercapnia reverses the protective effect of hypoxia, during infection. Thus, we report 1) that CA IX increases in the lungs of pneumonia rats and 2) that the CA IX IC domain and hypoxia promote PMVEC survival during infection.


Assuntos
Anidrase Carbônica IX/metabolismo , Células Endoteliais/enzimologia , Pulmão/enzimologia , Pneumonia Bacteriana/enzimologia , Infecções por Pseudomonas/enzimologia , Pseudomonas aeruginosa/metabolismo , Animais , Antígenos de Neoplasias/metabolismo , Hipóxia Celular , Humanos , Masculino , Ratos , Ratos Endogâmicos F344
4.
PLoS One ; 8(8): e71490, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23940764

RESUMO

Pulmonary artery endothelial cells (PAEC) in an intact vessel are continually exposed to serum, but unless injured, do not proliferate, constrained by confluence. In contrast, pulmonary artery smooth muscle cells (PASMC) attain, and maintain, confluence in the presence of minimal serum, protected from serum's stimulatory effects except when the endothelial barrier becomes more permeable. We hypothesized therefore, that confluent PASMC may be less constrained by contact inhibition in the presence of serum than PAEC and tested this idea by exposing confluent non-transformed human PAEC and PASMC to media containing increasing concentrations of fetal bovine serum (FBS) and determining cell growth over 7 days. PAEC that had attained confluence in low serum did not proliferate even when exposed to 5% serum, the highest concentration tested. In contrast, PASMC that attained confluence in low serum did proliferate once serum levels were increased, an effect that was dose dependent. Consistent with this observation, PASMC had more BrdU incorporation and a greater percentage of cells in S phase in 5% compared to 0.2% FBS, whereas no such difference was seen in PAEC. These results suggest that confluent human PAEC are resistant to the stimulatory effects of serum, whereas confluent PASMC can proliferate when serum levels are increased, an effect mediated in part by differences in phosphoinositide 3-kinase activation. This observation may be relevant to understanding the PASMC hyperplasia observed in humans and animals with pulmonary hypertension in which changes in endothelial permeability due to hypoxia or injury expose the underlying smooth muscle to serum.


Assuntos
Inibição de Contato , Músculo Liso Vascular/citologia , Miócitos de Músculo Liso/citologia , Artéria Pulmonar/citologia , Soro/fisiologia , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Inibição de Contato/efeitos dos fármacos , Meios de Cultura/farmacologia , Endotélio Vascular/citologia , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/fisiologia , Humanos , Músculo Liso Vascular/fisiologia , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/fisiologia , Proteína Oncogênica v-akt/metabolismo , Artéria Pulmonar/efeitos dos fármacos , Artéria Pulmonar/fisiologia , Proteína do Retinoblastoma/metabolismo
5.
Future Cardiol ; 7(2): 169-72, 2011 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-21453023

RESUMO

Evaluation of: Bauer EM, Qin Y, Miller TW et al.: Thrombospondin-1 supports blood pressure by limiting eNOS activation and endothelial dependent vasorelaxation. Cardiovasc. Res. 88, 471-481 (2010). Several lines of evidence, both in vivo and ex vivo, suggest that thrombospondin-1 (TSP-1) is important in maintaining systemic vascular tone. Recently published papers demonstrate that TSP-1 can inhibit vascular smooth muscle relaxation by interfering with the interaction between nitric oxide (NO) and soluble guanylyl cyclase, providing a possible mechanism of action to explain this observation. While these in vitro experiments in vascular smooth muscle cells were provocative, it is not clear how such a large protein circulating in the plasma could cross the intact endothelial basal membrane and regulate NO/cGMP signaling in smooth muscle in vivo. This raised the question of whether TSP-1 could modulate NO/cGMP signaling through another mechanism. Herein, we evaluate a recently published paper by Bauer and colleagues that examined whether TSP-1 could exert vasoactive effects without directly accessing the vascular smooth muscle. In their studies they found that TSP-1 could inhibit the NO/cGMP signaling pathway through an alternate mechanism: inhibiting the activation of endothelial NO synthase (eNOS), and therefore NO production in endothelial cells. These findings, combined with previous results from these investigators, suggest that TSP-1 can blunt NO/cGMP signaling through two different mechanisms: inhibiting NO production in endothelial cells by preventing the agonist-induced influx of Ca(2+) required to activate endothelial NO synthase and blunting the ability of endothelial-derived NO to activate soluble guanylyl cyclase in vascular smooth muscle cells. The importance of these two pathways in supporting systemic and pulmonary vascular tone in health and disease is unclear.

6.
Am J Physiol Lung Cell Mol Physiol ; 291(6): L1267-76, 2006 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-16861384

RESUMO

Although the accumulation of neutrophils in the lungs and airways is common to many inflammatory lung diseases, including acute lung injury, the alterations that neutrophils undergo as they leave the peripheral circulation and migrate into the lungs have not been well characterized. Human volunteers were exposed to endotoxin by bronchoscopic instillation. The resulting air space neutrophil accumulation and peripheral blood neutrophils were isolated 16 h later, compared with circulating neutrophils isolated before or after to the pulmonary endotoxin exposure, and compared with circulating neutrophils exposed to endotoxin in vitro. Microarray analysis was performed on air space, circulatory, and in vitro endotoxin-stimulated neutrophils. Functional analysis included the determination of neutrophil apoptosis, chemotaxis, release of cytokines and growth factors, and superoxide anion release. Dramatic gene expression differences were apparent between air space and circulating neutrophils: approximately 15% of expressed genes have altered expression levels, including broad increases in inflammatory- and chemotaxis-related genes, as well as antiapoptotic and IKK-activating pathways. Functional analysis of air space compared with circulating neutrophils showed increased superoxide release, diminished apoptosis, decreased IL-8-induced chemotaxis, and a pattern of IL-8, macrophage inflammatory protein-1beta, monocyte chemoattractant protein-1, and tumor necrosis factor-alpha release different from either unstimulated or LPS-stimulated circulating neutrophils. Many of these changes are not elicited by in vitro treatment with endotoxin. Limited differences were detected between circulating neutrophils isolated before and 16 h after pulmonary endotoxin instillation. These results suggest that neutrophils sequestered in the lung become fundamentally different from those resident in the circulation, and this difference is distinct from in vitro activation with endotoxin.


Assuntos
Citocinas/genética , Genômica , Macrófagos Alveolares/fisiologia , Neutrófilos/fisiologia , Movimento Celular , Quimiotaxia de Leucócito , Regulação da Expressão Gênica , Humanos , Cinética , Análise de Sequência com Séries de Oligonucleotídeos
7.
Am J Physiol Lung Cell Mol Physiol ; 290(6): L1111-6, 2006 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-16399787

RESUMO

Growth and development of the lung normally occur in the low oxygen environment of the fetus. The role of this low oxygen environment on fetal lung endothelial cell growth and function is unknown. We hypothesized that low oxygen tension during fetal life enhances pulmonary artery endothelial cell (PAEC) growth and function and that nitric oxide (NO) production modulates fetal PAEC responses to low oxygen tension. To test this hypothesis, we compared the effects of fetal (3%) and room air (RA) oxygen tension on fetal PAEC growth, proliferation, tube formation, and migration in the presence and absence of the NO synthase (NOS) inhibitor N(omega)-nitro-l-arginine (LNA), and an NO donor, S-nitroso-N-acetylpenicillamine (SNAP). Compared with fetal PAEC grown in RA, 3% O(2) increased tube formation by over twofold (P < 0.01). LNA treatment reduced tube formation in 3% O(2) but had no affect on tube formation in RA. Treatment with SNAP increased tube formation during RA exposure to levels observed in 3% O(2). Exposure to 3% O(2) for 48 h attenuated cell number (by 56%), and treatment with LNA reduced PAEC growth by 44% in both RA and 3% O(2). We conclude that low oxygen tension enhances fetal PAEC tube formation and that NO is essential for normal PAEC growth, migration, and tube formation. Furthermore, we conclude that in fetal cells exposed to the relative hyperoxia of RA, 21% O(2), NO overcomes the inhibitory effects of the increased oxygen, allowing normal PAEC angiogenesis and branching. We speculate that NO production maintains intrauterine lung vascular growth and development during exposure to low O(2) in the normal fetus. We further speculate that NO is essential for pulmonary angiogenesis in fetal animal exposed to increased oxygen tension of RA and that impaired endothelial NO production may contribute to the abnormalities of angiogenesis see in infants with bronchopulmonary dysplasia.


Assuntos
Endotélio Vascular/fisiologia , Neovascularização Fisiológica/efeitos dos fármacos , Óxido Nítrico/farmacologia , Artéria Pulmonar/fisiologia , Animais , Divisão Celular/efeitos dos fármacos , Endotélio Vascular/efeitos dos fármacos , Feminino , Hipóxia , Técnicas In Vitro , Doadores de Óxido Nítrico/farmacologia , Óxido Nítrico Sintase/antagonistas & inibidores , Oxigênio/farmacologia , Gravidez , Artéria Pulmonar/embriologia , S-Nitroso-N-Acetilpenicilamina/farmacologia , Ovinos
9.
Blood ; 104(13): 3878-85, 2004 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-15339848

RESUMO

Recombinant human activated protein C (rhAPC) is a natural anticoagulant with potentially important anti-inflammatory properties. In humans with severe sepsis, rhAPC treatment reduces mortality, but mechanisms responsible have not been well characterized. Accumulation of activated neutrophils in the lungs and other organs during severe infection contributes to sepsis-induced organ dysfunction, including acute inflammatory lung injury. Because neutrophils express an APC receptor, we hypothesized that immunomodulatory effects of rhAPC occur, in part, via modulation of neutrophil responses. To examine this issue, we performed a double-blinded, placebo-controlled study of rhAPC in a human model of endotoxin-induced pulmonary inflammation. Administration of rhAPC significantly reduced leukocyte accumulation to the airspaces, independent of pulmonary cytokine or chemokine release. Neutrophils recovered from bronchoalveolar lavage fluid of volunteers receiving rhAPC demonstrated decreased chemotaxis ex vivo. Decreased neutrophil chemotaxis following exposure to rhAPC was confirmed in vitro. No differences were detected in gene expression, kinase activation, cytokine release, cell survival, or apoptosis of neutrophils recovered in the presence or absence of rhAPC. These studies demonstrate that rhAPC reduces both endotoxin-induced accumulation of leukocytes in the airspaces and neutrophil chemotaxis. These rhAPC-induced effects on neutrophil function may represent a mechanism by which rhAPC improves survival in patients with sepsis.


Assuntos
Quimiotaxia de Leucócito/efeitos dos fármacos , Pneumopatias/tratamento farmacológico , Neutrófilos/fisiologia , Proteína C/uso terapêutico , Adolescente , Adulto , Método Duplo-Cego , Endotoxinas/toxicidade , Feminino , Humanos , Masculino , Neutrófilos/efeitos dos fármacos , Proteínas Recombinantes/uso terapêutico , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
10.
Circ Res ; 92(5): 501-9, 2003 Mar 21.
Artigo em Inglês | MEDLINE | ID: mdl-12600884

RESUMO

Advanced pulmonary arterial hypertension is characterized by extensive vascular remodeling that is usually resistant to vasodilator therapy. Mevastatin is an inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, the rate-limiting step for cholesterol synthesis. HMG-CoA reductase inhibitors have been shown to upregulate the cyclin-dependent kinase inhibitor p27Kip1 and to block cell proliferation through cholesterol-independent pathways. The aim of this study was to determine the effect of mevastatin on DNA synthesis, cell cycle progression, and cell proliferation in rat pulmonary artery smooth muscle cells (PASMCs). We found that mevastatin induced G1 arrest and decreased DNA synthesis in rat PASMCs and did so in association with an increase in both total and cyclin E-bound p27Kip1. This caused a marked decrease in cyclin E kinase activity, which suggests an important role for p27Kip1 in the ability of mevastatin to induce G1 arrest. However, in PASMCs lacking functional p27Kip1, mevastatin still decreased cyclin E kinase activity, caused G1 arrest, and decreased DNA synthesis. In p27Kip1-deficient PASMCs, mevastatin induced a greater reduction of cyclin E protein levels (to 35% of control) than in wild-type cells (to 70% of control) and also reduced the phosphorylation of cdk2 on threonine 160. Mevastatin also caused apoptosis in both wild-type and p27Kip1-deficient PASMCs and was able to do so at a dose that did not induce cell cycle arrest. These data suggest that HMG-CoA reductase inhibitors can both inhibit cell proliferation and induce apoptosis in PASMCs through p27Kip1-independent pathways and may be important therapeutic agents in pulmonary arterial hypertension.


Assuntos
Apoptose , Proteínas de Ciclo Celular/fisiologia , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Lovastatina/análogos & derivados , Lovastatina/farmacologia , Músculo Liso Vascular/metabolismo , Artéria Pulmonar/citologia , Proteínas Supressoras de Tumor/fisiologia , Animais , Proteínas de Ciclo Celular/genética , Células Cultivadas , Meios de Cultura , Ciclina E/metabolismo , Inibidor de Quinase Dependente de Ciclina p27 , Quinases Ciclina-Dependentes/metabolismo , DNA/biossíntese , Fase G1 , Cinética , Masculino , Camundongos , Músculo Liso Vascular/citologia , Músculo Liso Vascular/efeitos dos fármacos , Mutação , Ratos , Ratos Sprague-Dawley , Transdução de Sinais , Proteínas Supressoras de Tumor/genética
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