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1.
Circulation ; 2024 Jun 14.
Artículo en Inglés | MEDLINE | ID: mdl-38873770

RESUMEN

BACKGROUND: Endothelial cell (EC) apoptosis and proliferation of apoptosis-resistant cells is a hallmark of pulmonary hypertension (PH). Yet, why some ECs die and others proliferate and how this contributes to vascular remodeling is unclear. We hypothesized that this differential response may: (1) relate to different EC subsets, namely pulmonary artery (PAECs) versus microvascular ECs (MVECs); (2) be attributable to autophagic activation in both EC subtypes; and (3) cause replacement of MVECs by PAECs with subsequent distal vessel muscularization. METHODS: EC subset responses to chronic hypoxia were assessed by single-cell RNA sequencing of murine lungs. Proliferative versus apoptotic responses, activation, and role of autophagy were assessed in human and rat PAECs and MVECs, and in precision-cut lung slices of wild-type mice or mice with endothelial deficiency in the autophagy gene Atg7 (Atg7EN-KO). Abundance of PAECs versus MVECs in precapillary microvessels was assessed in lung tissue from patients with PH and animal models on the basis of structural or surface markers. RESULTS: In vitro and in vivo, PAECs proliferated in response to hypoxia, whereas MVECs underwent apoptosis. Single-cell RNA sequencing analyses support these findings in that hypoxia induced an antiapoptotic, proliferative phenotype in arterial ECs, whereas capillary ECs showed a propensity for cell death. These distinct responses were prevented in hypoxic Atg7EN-KO mice or after ATG7 silencing, yet replicated by autophagy stimulation. In lung tissue from mice, rats, or patients with PH, the abundance of PAECs in precapillary arterioles was increased, and that of MVECs reduced relative to controls, indicating replacement of microvascular by macrovascular ECs. EC replacement was prevented by genetic or pharmacological inhibition of autophagy in vivo. Conditioned medium from hypoxic PAECs yet not MVECs promoted pulmonary artery smooth muscle cell proliferation and migration in a platelet-derived growth factor-dependent manner. Autophagy inhibition attenuated PH development and distal vessel muscularization in preclinical models. CONCLUSIONS: Autophagic activation by hypoxia induces in parallel PAEC proliferation and MVEC apoptosis. These differential responses cause a progressive replacement of MVECs by PAECs in precapillary pulmonary arterioles, thus providing a macrovascular context that in turn promotes pulmonary artery smooth muscle cell proliferation and migration, ultimately driving distal vessel muscularization and the development of PH.

2.
Eur Respir J ; 60(2)2022 08.
Artículo en Inglés | MEDLINE | ID: mdl-35058252

RESUMEN

BACKGROUND: Cigarette smokers are at increased risk of acquiring influenza, developing severe disease and requiring hospitalisation/intensive care unit admission following infection. However, immune mechanisms underlying this predisposition are incompletely understood, and therapeutic strategies for influenza are limited. METHODS: We used a mouse model of concurrent cigarette smoke exposure and H1N1 influenza infection, colony-stimulating factor (CSF)3 supplementation/receptor (CSF3R) blockade and single-cell RNA sequencing (scRNAseq) to investigate this relationship. RESULTS: Cigarette smoke exposure exacerbated features of viral pneumonia such as oedema, hypoxaemia and pulmonary neutrophilia. Smoke-exposed infected mice demonstrated an increase in viral (v)RNA, but not replication-competent viral particles, relative to infection-only controls. Interstitial rather than airspace neutrophilia positively predicted morbidity in smoke-exposed infected mice. Screening of pulmonary cytokines using a novel dysregulation score identified an exacerbated expression of CSF3 and interleukin-6 in the context of smoke exposure and influenza. Recombinant (r)CSF3 supplementation during influenza aggravated morbidity, hypothermia and oedema, while anti-CSF3R treatment of smoke-exposed infected mice improved alveolar-capillary barrier function. scRNAseq delineated a shift in the distribution of Csf3 + cells towards neutrophils in the context of cigarette smoke and influenza. However, although smoke-exposed lungs were enriched for infected, highly activated neutrophils, gene signatures of these cells largely reflected an exacerbated form of typical influenza with select unique regulatory features. CONCLUSION: This work provides novel insight into the mechanisms by which cigarette smoke exacerbates influenza infection, unveiling potential therapeutic targets (e.g. excess vRNA accumulation, oedematous CSF3R signalling) for use in this context, and potential limitations for clinical rCSF3 therapy during viral infectious disease.


Asunto(s)
Fumar Cigarrillos , Subtipo H1N1 del Virus de la Influenza A , Gripe Humana , Animales , Fumar Cigarrillos/efectos adversos , Humanos , Pulmón/metabolismo , Ratones , Ratones Endogámicos C57BL , Neutrófilos , Nicotiana
3.
Microvasc Res ; 139: 104259, 2022 01.
Artículo en Inglés | MEDLINE | ID: mdl-34624307

RESUMEN

Blood flow pulsatility is an important determinant of macro- and microvascular physiology. Pulsatility is damped largely in the microcirculation, but the characteristics of this damping and the factors that regulate it have not been fully elucidated yet. Applying computational approaches to real microvascular network geometry, we examined the pattern of pulsatility damping and the role of potential damping factors, including pulse frequency, vascular viscous resistance, vascular compliance, viscoelastic behavior of the vessel wall, and wave propagation and reflection. To this end, three full rat mesenteric vascular networks were reconstructed from intravital microscopic recordings, a one-dimensional (1D) model was used to reproduce pulsatile properties within the network, and potential damping factors were examined by sensitivity analysis. Results demonstrate that blood flow pulsatility is predominantly damped at the arteriolar side and remains at a low level at the venular side. Damping was sensitive to pulse frequency, vascular viscous resistance and vascular compliance, whereas viscoelasticity of the vessel wall or wave propagation and reflection contributed little to pulsatility damping. The present results contribute to our understanding of mechanical forces and their regulation in the microcirculation.


Asunto(s)
Arteriolas/fisiología , Mesenterio/irrigación sanguínea , Microcirculación , Modelos Cardiovasculares , Flujo Pulsátil , Circulación Esplácnica , Vénulas/fisiología , Animales , Microscopía Intravital , Masculino , Ratas Wistar , Estrés Mecánico , Factores de Tiempo , Resistencia Vascular
4.
Sci Rep ; 11(1): 5265, 2021 03 04.
Artículo en Inglés | MEDLINE | ID: mdl-33664277

RESUMEN

Mesenchymal stem/stromal cells (MSCs) have demonstrated efficacy in pre-clinical models of inflammation and tissue injury, including in models of lung injury and infection. Rolling, adhesion and transmigration of MSCs appears to play a role during MSC kinetics in the systemic vasculature. However, a large proportion of MSCs become entrapped within the lungs after intravenous administration, while the initial kinetics and the site of arrest of MSCs in the pulmonary vasculature are unknown. We examined the kinetics of intravascularly administered MSCs in the pulmonary vasculature using a microfluidic system in vitro and intra-vital microscopy of intact mouse lung. In vitro, MSCs bound to endothelium under static conditions but not under laminar flow. VCAM-1 antibodies did not affect MSC binding. Intravital microscopy demonstrated MSC arrest at pulmonary micro-vessel bifurcations due to size obstruction. Retention of MSCs in the pulmonary microvasculature was increased in Escherichia coli-infected animals. Trapped MSCs deformed over time and appeared to release microvesicles. Labelled MSCs retained therapeutic efficacy against pneumonia. Our results suggest that MSCs are physically obstructed in pulmonary vasculature and do not display properties of rolling/adhesion, while retention of MSCs in the infected lung may require receptor interaction.


Asunto(s)
Vasos Sanguíneos/trasplante , Pulmón/diagnóstico por imagen , Trasplante de Células Madre Mesenquimatosas , Neumonía/terapia , Administración Intravenosa , Animales , Vasos Sanguíneos/diagnóstico por imagen , Vasos Sanguíneos/patología , Sistema Cardiovascular/metabolismo , Modelos Animales de Enfermedad , Humanos , Cinética , Pulmón/irrigación sanguínea , Pulmón/metabolismo , Pulmón/patología , Células Madre Mesenquimatosas/citología , Ratones , Neumonía/diagnóstico por imagen , Neumonía/metabolismo , Neumonía/patología
5.
Blood ; 137(5): 690-701, 2021 02 04.
Artículo en Inglés | MEDLINE | ID: mdl-33232973

RESUMEN

Transfusion-related acute lung injury (TRALI) is a hazardous transfusion complication with an associated mortality of 5% to 15%. We previously showed that stored (5 days) but not fresh platelets (1 day) cause TRALI via ceramide-mediated endothelial barrier dysfunction. As biological ceramides are hydrophobic, extracellular vesicles (EVs) may be required to shuttle these sphingolipids from platelets to endothelial cells. Adding to complexity, EV formation in turn requires ceramide. We hypothesized that ceramide-dependent EV formation from stored platelets and EV-dependent sphingolipid shuttling induces TRALI. EVs formed during storage of murine platelets were enumerated, characterized for sphingolipids, and applied in a murine TRALI model in vivo and for endothelial barrier assessment in vitro. Five-day EVs were more abundant, had higher long-chain ceramide (C16:0, C18:0, C20:0), and lower sphingosine-1-phosphate (S1P) content than 1-day EVs. Transfusion of 5-day, but not 1-day, EVs induced characteristic signs of lung injury in vivo and endothelial barrier disruption in vitro. Inhibition or supplementation of ceramide-forming sphingomyelinase reduced or enhanced the formation of EVs, respectively, but did not alter the injuriousness per individual EV. Barrier failure was attenuated when EVs were abundant in or supplemented with S1P. Stored human platelet 4-day EVs were more numerous compared with 2-day EVs, contained more long-chain ceramide and less S1P, and caused more endothelial cell barrier leak. Hence, platelet-derived EVs become more numerous and more injurious (more long-chain ceramide, less S1P) during storage. Blockade of sphingomyelinase, EV elimination, or supplementation of S1P during platelet storage may present promising strategies for TRALI prevention.


Asunto(s)
Vesículas Extracelulares/fisiología , Transfusión de Plaquetas/efectos adversos , Esfingolípidos/metabolismo , Lesión Pulmonar Aguda Postransfusional/etiología , Animales , Plaquetas/ultraestructura , Conservación de la Sangre , Ceramidas/metabolismo , Células Endoteliales/fisiología , Endotoxinas/toxicidad , Humanos , Lisofosfolípidos/fisiología , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Modelos Biológicos , Esfingomielina Fosfodiesterasa/antagonistas & inhibidores , Esfingomielina Fosfodiesterasa/deficiencia , Esfingomielina Fosfodiesterasa/fisiología , Esfingosina/análogos & derivados , Esfingosina/fisiología , Lesión Pulmonar Aguda Postransfusional/metabolismo , Lesión Pulmonar Aguda Postransfusional/prevención & control
6.
Front Physiol ; 11: 291, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32308629

RESUMEN

Intravital microscopy (IVM) offers unique possibilities for the observation of biological processes and disease related mechanisms in vivo. Especially for anatomically complex and dynamic organs such as the lung and its main functional unit, the alveolus, IVM provides exclusive advantages in terms of spatial and temporal resolution. By the use of lung windows, which have advanced and improved over time, direct access to the lung surface is provided. In this review we will discuss two main topics, namely alveolar dynamics and perfusion from the perspective of IVM-based studies. Of special interest are unanswered questions regarding alveolar dynamics such as: What are physiologic alveolar dynamics? How do these dynamics change under pathologic conditions and how do those changes contribute to ventilator-induced lung injury? How can alveolar dynamics be targeted in a beneficial way? With respect to alveolar perfusion IVM has propelled our understanding of the pulmonary microcirculation and its perfusion, as well as pulmonary vasoreactivity, permeability and immunological aspects. Whereas the general mechanism behind these processes are understood, we still lack a proper understanding of the complex, multidimensional interplay between alveolar ventilation and microvascular perfusion, capillary recruitment, or vascular immune responses under physiologic and pathologic conditions. These are only part of the unanswered questions and problems, which we still have to overcome. IVM as the tool of choice might allow us to answer part of these questions within the next years or decades. As every method, IVM has advantages as well as limitations, which have to be taken into account for data analysis and interpretation, which will be addressed in this review.

7.
Hypertension ; 74(2): 295-304, 2019 08.
Artículo en Inglés | MEDLINE | ID: mdl-31291149

RESUMEN

Subendocardial damage is among the first cardiac manifestations of hypertension and is already present in asymptomatic disease states. Accordingly, markers of subendocardial impairment may facilitate early detection of cardiac damages and risk stratification under these conditions. This study aimed to investigate the impact of subendocardial damage on myocardial microstructure and function to elucidate early pathophysiologic processes and to identify corresponding diagnostic measures. Mice (n=38) were injected with isoproterenol to induce isolated subendocardial scarring or saline as corresponding control. Cardiac function and myocardial deformation were determined by high-frequency echocardiography. The cardiac stress response was assessed in a graded exercise test and during dobutamine stress echocardiography. Myocardial microstructure was studied ex vivo by 7 T diffusion tensor magnetic resonance imaging at a spatial resolution of 100×100×100 µm 3 . Results were correlated with histology and biomarker expression. Subendocardial fibrosis was accompanied by diastolic dysfunction, impaired longitudinal deformation (global peak longitudinal strain [LS]: -12.5±0.5% versus -15.6±0.5%; P<0.001) and elevated biomarker expression (ANP [atrial natriuretic peptide], Galectin-3, and ST2). Systolic function and cardiac stress response remained preserved. Diffusion tensor magnetic resonance imaging revealed a left-shift in helix angle towards lower values in isoproterenol-treated animals, which was mainly determined by subepicardial myofibers (mean helix angle: 2.2±0.8° versus 5.9±1.0°; P<0.01). Longitudinal strain and subepicardial helix angle were highly predictive for subendocardial fibrosis (sensitivity, 82%-92% and specificity, 89%-90%). The results indicate that circumscribed subendocardial damage alone can cause several hallmarks observed in cardiovascular high-risk patients. Microstructural remodeling under these conditions involves also remote regions, and corresponding changes in longitudinal strain and helix angle might serve as diagnostic markers.


Asunto(s)
Endocardio/patología , Interpretación de Imagen Asistida por Computador , Isoproterenol/efectos adversos , Imagen por Resonancia Cinemagnética/métodos , Disfunción Ventricular Izquierda/diagnóstico por imagen , Animales , Biopsia con Aguja , Modelos Animales de Enfermedad , Ecocardiografía/métodos , Endocardio/diagnóstico por imagen , Endocardio/lesiones , Fibrosis/diagnóstico por imagen , Fibrosis/patología , Alemania , Humanos , Inmunohistoquímica , Inyecciones Subcutáneas , Isoproterenol/administración & dosificación , Modelos Lineales , Ratones , Ratones Endogámicos , Curva ROC , Distribución Aleatoria , Valores de Referencia , Volumen Sistólico/fisiología , Análisis de Supervivencia , Disfunción Ventricular Izquierda/patología
8.
Intensive Care Med Exp ; 7(Suppl 1): 34, 2019 Jul 25.
Artículo en Inglés | MEDLINE | ID: mdl-31346797

RESUMEN

Mechanical ventilation is a life-saving therapy in patients with acute respiratory distress syndrome (ARDS). However, mechanical ventilation itself causes severe co-morbidities in that it can trigger ventilator-associated lung injury (VALI) in humans or ventilator-induced lung injury (VILI) in experimental animal models. Therefore, optimization of ventilation strategies is paramount for the effective therapy of critical care patients. A major problem in the stratification of critical care patients for personalized ventilation settings, but even more so for our overall understanding of VILI, lies in our limited insight into the effects of mechanical ventilation at the actual site of injury, i.e., the alveolar unit. Unfortunately, global lung mechanics provide for a poor surrogate of alveolar dynamics and methods for the in-depth analysis of alveolar dynamics on the level of individual alveoli are sparse and afflicted by important limitations. With alveolar dynamics in the intact lung remaining largely a "black box," our insight into the mechanisms of VALI and VILI and the effectiveness of optimized ventilation strategies is confined to indirect parameters and endpoints of lung injury and mortality.In the present review, we discuss emerging concepts of alveolar dynamics including alveolar expansion/contraction, stability/instability, and opening/collapse. Many of these concepts remain still controversial, in part due to limitations of the different methodologies applied. We therefore preface our review with an overview of existing technologies and approaches for the analysis of alveolar dynamics, highlighting their individual strengths and limitations which may provide for a better appreciation of the sometimes diverging findings and interpretations. Joint efforts combining key technologies in identical models to overcome the limitations inherent to individual methodologies are needed not only to provide conclusive insights into lung physiology and alveolar dynamics, but ultimately to guide critical care patient therapy.

9.
Pulm Circ ; 8(2): 2045893218757596, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-29480134

RESUMEN

While pulmonary hypertension (PH) has traditionally not been considered as a disease that is directly linked to or, potentially, even caused by inflammation, a rapidly growing body of evidence has demonstrated the accumulation of a variety of inflammatory and immune cells in PH lungs, in and around the wall of remodeled pulmonary resistance vessels and in the vicinity of plexiform lesions, respectively. Concomitantly, abundant production and release of various inflammatory mediators has been documented in both PH patients and experimental models of PH. While these findings unequivocally demonstrate an inflammatory component in PH, they have fueled an intense and presently ongoing debate as to the nature of this inflammatory aspect: is it a mere bystander of or response to the actual disease process, or is it a pathomechanistic contributor or potentially even a trigger of endothelial injury, smooth muscle hypertrophy and hyperplasia, and the resulting lung vascular remodeling? In this review, we will discuss the present evidence for an inflammatory component in PH disease with a specific focus on the potential role of the endothelium in this scenario and highlight future avenues of experimental investigation which may lead to novel therapeutic interventions.

10.
Am J Physiol Lung Cell Mol Physiol ; 312(5): L625-L637, 2017 05 01.
Artículo en Inglés | MEDLINE | ID: mdl-28283474

RESUMEN

Pulmonary complications from stored blood products are the leading cause of mortality related to transfusion. Transfusion-related acute lung injury is mediated by antibodies or bioactive mediators, yet underlying mechanisms are incompletely understood. Sphingolipids such as ceramide regulate lung injury, and their composition changes as a function of time in stored blood. Here, we tested the hypothesis that aged platelets may induce lung injury via a sphingolipid-mediated mechanism. To assess this hypothesis, a two-hit mouse model was devised. Recipient mice were treated with 2 mg/kg intraperitoneal lipopolysaccharide (priming) 2 h before transfusion of 10 ml/kg stored (1-5 days) platelets treated with or without addition of acid sphingomyelinase inhibitor ARC39 or platelets from acid sphingomyelinase-deficient mice, which both reduce ceramide formation. Transfused mice were examined for signs of pulmonary neutrophil accumulation, endothelial barrier dysfunction, and histological evidence of lung injury. Sphingolipid profiles in stored platelets were analyzed by mass spectrophotometry. Transfusion of aged platelets into primed mice induced characteristic features of lung injury, which increased in severity as a function of storage time. Ceramide accumulated in platelets during storage, but this was attenuated by ARC39 or in acid sphingomyelinase-deficient platelets. Compared with wild-type platelets, transfusion of ARC39-treated or acid sphingomyelinase-deficient aged platelets alleviated lung injury. Aged platelets elicit lung injury in primed recipient mice, which can be alleviated by pharmacological inhibition or genetic deletion of acid sphingomyelinase. Interventions targeting sphingolipid formation represent a promising strategy to increase the safety and longevity of stored blood products.


Asunto(s)
Lesión Pulmonar Aguda/enzimología , Lesión Pulmonar Aguda/etiología , Plaquetas/metabolismo , Senescencia Celular , Transfusión de Plaquetas/efectos adversos , Esfingomielina Fosfodiesterasa/metabolismo , Lesión Pulmonar Aguda/patología , Animales , Plaquetas/efectos de los fármacos , Ceramidas/metabolismo , Inhibidores Enzimáticos/farmacología , Eliminación de Gen , Humanos , Inflamación/patología , Lipopolisacáridos/farmacología , Macrófagos/patología , Masculino , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Esfingomielina Fosfodiesterasa/antagonistas & inhibidores , Esfingomielina Fosfodiesterasa/deficiencia , Factores de Tiempo
11.
Am J Physiol Lung Cell Mol Physiol ; 312(5): L710-L721, 2017 05 01.
Artículo en Inglés | MEDLINE | ID: mdl-28235950

RESUMEN

Over past years, a critical role for the immune system and, in particular, for mast cells in the pathogenesis of pulmonary hypertension (PH) has emerged. However, the way in which mast cells promote PH is still poorly understood. Here, we investigated the mechanisms by which mast cells may contribute to PH, specifically focusing on the interaction between the innate and adaptive immune response and the role of B cells and autoimmunity. Experiments were performed in Sprague-Dawley rats and B cell-deficient JH-KO rats in the monocrotaline, Sugen/hypoxia, and the aortic banding model of PH. Hemodynamics, cell infiltration, IL-6 expression, and vascular remodeling were analyzed. Gene array analyses revealed constituents of immunoglobulins as most prominently regulated mast cell-dependent genes in the lung in experimental PH. IL-6 was shown to link mast cells to B cells, as 1) IL-6 was upregulated and colocalized with mast cells and was reduced by mast-cell stabilizers and 2) IL-6 or mast cell blockade reduced B cells in lungs of monocrotaline-treated rats. A functional role for B cells in PH was demonstrated in that either blocking B cells by an anti-CD20 antibody or B-cell deficiency in JH-KO rats attenuated right ventricular systolic pressure and vascular remodeling in experimental PH. We here identify a mast cell-B cell axis driven by IL-6 as a critical immune pathway in the pathophysiology of PH. Our results provide novel insights into the role of the immune system in PH, which may be therapeutically exploited by targeted immunotherapy.


Asunto(s)
Linfocitos B/metabolismo , Hipertensión Pulmonar/fisiopatología , Pulmón/irrigación sanguínea , Pulmón/fisiopatología , Mastocitos/metabolismo , Remodelación Vascular , Animales , Autoanticuerpos/metabolismo , Presión Sanguínea , Ventrículos Cardíacos/patología , Ventrículos Cardíacos/fisiopatología , Interleucina-6/metabolismo , Masculino , Modelos Biológicos , Análisis de Secuencia por Matrices de Oligonucleótidos , Ratas Sprague-Dawley , Sístole , Factores de Tiempo
12.
Blood ; 129(18): 2557-2569, 2017 05 04.
Artículo en Inglés | MEDLINE | ID: mdl-28202460

RESUMEN

Transfusion-related acute lung injury (TRALI) is the leading cause of transfusion-related fatalities and is characterized by acute respiratory distress following blood transfusion. Donor antibodies are frequently involved; however, the pathogenesis and protective mechanisms in the recipient are poorly understood, and specific therapies are lacking. Using newly developed murine TRALI models based on injection of anti-major histocompatibility complex class I antibodies, we found CD4+CD25+FoxP3+ T regulatory cells (Tregs) and CD11c+ dendritic cells (DCs) to be critical effectors that protect against TRALI. Treg or DC depletion in vivo resulted in aggravated antibody-mediated acute lung injury within 90 minutes with 60% mortality upon DC depletion. In addition, resistance to antibody-mediated TRALI was associated with increased interleukin-10 (IL-10) levels, and IL-10 levels were found to be decreased in mice suffering from TRALI. Importantly, IL-10 injection completely prevented and rescued the development of TRALI in mice and may prove to be a promising new therapeutic approach for alleviating lung injury in this serious complication of transfusion.


Asunto(s)
Lesión Pulmonar Aguda , Células Dendríticas/inmunología , Interleucina-10 , Linfocitos T Reguladores/inmunología , Reacción a la Transfusión , Lesión Pulmonar Aguda/etiología , Lesión Pulmonar Aguda/inmunología , Lesión Pulmonar Aguda/prevención & control , Animales , Anticuerpos/inmunología , Células Dendríticas/patología , Interleucina-10/inmunología , Interleucina-10/farmacología , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Linfocitos T Reguladores/patología
13.
Am J Respir Crit Care Med ; 193(4): 396-406, 2016 Feb 15.
Artículo en Inglés | MEDLINE | ID: mdl-26513710

RESUMEN

RATIONALE: Improved ventilation strategies have been the mainstay for reducing mortality in acute respiratory distress syndrome. Their unique clinical effectiveness is, however, unmatched by our understanding of the underlying mechanobiology, and their impact on alveolar dynamics and gas exchange remains largely speculative. OBJECTIVES: To assess changes in alveolar dynamics and associated effects on local gas exchange in experimental models of acute lung injury (ALI) and their responsiveness to sighs. METHODS: Alveolar dynamics and local gas exchange were studied in vivo by darkfield microscopy and multispectral oximetry in experimental murine models of ALI induced by hydrochloric acid, Tween instillation, or in antibody-mediated transfusion-related ALI. MEASUREMENTS AND MAIN RESULTS: Independent of injury mode, ALI resulted in asynchronous alveolar ventilation characteristic of alveolar pendelluft, which either spontaneously resolved or progressed to a complete cessation or even inversion of alveolar ventilation. The functional relevance of the latter phenomena was evident as impaired blood oxygenation in juxtaposed lung capillaries. Individual sighs (2 × 10 s at inspiratory plateau pressure of 30 cm H2O) largely restored normal alveolar dynamics and gas exchange in acid-induced ALI, yet not in Tween-induced surfactant depletion. CONCLUSIONS: We describe for the first time in detail the different forms and temporal sequence of impaired alveolar dynamics in the acutely injured lung and report the first direct visualization of alveolar pendelluft. Moreover, we identify individual sighs as an effective strategy to restore intact alveolar ventilation by a mechanism independent of alveolar collapse and reopening.


Asunto(s)
Lesión Pulmonar Aguda/terapia , Alveolos Pulmonares/fisiopatología , Mecánica Respiratoria/fisiología , Animales , Modelos Animales de Enfermedad , Espiración/fisiología , Masculino , Ratones , Ratones Endogámicos BALB C , Oximetría
14.
Am J Respir Cell Mol Biol ; 54(3): 370-83, 2016 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-26222277

RESUMEN

The cation channel transient receptor potential vanilloid (TRPV) 4 is expressed in endothelial and immune cells; however, its role in acute lung injury (ALI) is unclear. The functional relevance of TRPV4 was assessed in vivo, in isolated murine lungs, and in isolated neutrophils. Genetic deficiency of TRPV4 attenuated the functional, histological, and inflammatory hallmarks of acid-induced ALI. Similar protection was obtained with prophylactic administration of the TRPV4 inhibitor, GSK2193874; however, therapeutic administration of the TRPV4 inhibitor, HC-067047, after ALI induction had no beneficial effect. In isolated lungs, platelet-activating factor (PAF) increased vascular permeability in lungs perfused with trpv4(+/+) more than with trpv4(-/-) blood, independent of lung genotype, suggesting a contribution of TRPV4 on blood cells to lung vascular barrier failure. In neutrophils, TRPV4 inhibition or deficiency attenuated the PAF-induced increase in intracellular calcium. PAF induced formation of epoxyeicosatrienoic acids by neutrophils, which, in turn, stimulated TRPV4-dependent Ca(2+) signaling, whereas inhibition of epoxyeicosatrienoic acid formation inhibited the Ca(2+) response to PAF. TRPV4 deficiency prevented neutrophil responses to proinflammatory stimuli, including the formation of reactive oxygen species, neutrophil adhesion, and chemotaxis, putatively due to reduced activation of Rac. In chimeric mice, however, the majority of protective effects in acid-induced ALI were attributable to genetic deficiency of TRPV4 in parenchymal tissue, whereas TRPV4 deficiency in circulating blood cells primarily reduced lung myeloperoxidase activity. Our findings identify TRPV4 as novel regulator of neutrophil activation and suggest contributions of both parenchymal and neutrophilic TRPV4 in the pathophysiology of ALI.


Asunto(s)
Lesión Pulmonar Aguda/metabolismo , Pulmón/metabolismo , Activación Neutrófila , Neutrófilos/metabolismo , Canales Catiónicos TRPV/metabolismo , Lesión Pulmonar Aguda/inducido químicamente , Lesión Pulmonar Aguda/genética , Lesión Pulmonar Aguda/prevención & control , Animales , Trasplante de Médula Ósea , Señalización del Calcio , Permeabilidad Capilar , Modelos Animales de Enfermedad , Humanos , Ácido Clorhídrico , Pulmón/irrigación sanguínea , Pulmón/efectos de los fármacos , Masculino , Ratones Noqueados , Morfolinas/farmacología , Activación Neutrófila/efectos de los fármacos , Neutrófilos/efectos de los fármacos , Neumonía/metabolismo , Edema Pulmonar/metabolismo , Pirroles/farmacología , Canales Catiónicos TRPV/antagonistas & inhibidores , Canales Catiónicos TRPV/deficiencia , Canales Catiónicos TRPV/genética
15.
Sci Rep ; 5: 11030, 2015 Jun 05.
Artículo en Inglés | MEDLINE | ID: mdl-26046800

RESUMEN

Seasonal influenza virus infections cause hundreds of thousands of deaths annually while viral mutation raises the threat of a novel pandemic strain. Antiviral drugs exhibit limited efficacy unless administered early and may induce viral resistance. Thus, targeting the host response directly has been proposed as a novel therapeutic strategy with the added potential benefit of not eliciting viral resistance. Severe influenza virus infections are complicated by respiratory failure due to the development of lung microvascular leak and acute lung injury. We hypothesized that enhancing lung endothelial barrier integrity could improve the outcome. Here we demonstrate that the Tie2-agonist tetrameric peptide Vasculotide improves survival in murine models of severe influenza, even if administered as late as 72 hours after infection; the benefit was observed using three strains of the virus and two strains of mice. The effect required Tie2, was independent of viral replication and did not impair lung neutrophil recruitment. Administration of the drug decreased lung edema, arterial hypoxemia and lung endothelial apoptosis; importantly, Vasculotide is inexpensive to produce, is chemically stable and is unrelated to any Tie2 ligands. Thus, Vasculotide may represent a novel and practical therapy for severe infections with influenza.


Asunto(s)
Infecciones por Orthomyxoviridae/tratamiento farmacológico , Péptidos/uso terapéutico , Receptor TIE-2/agonistas , Animales , Líquido del Lavado Bronquioalveolar/citología , Células Cultivadas , Modelos Animales de Enfermedad , Células Endoteliales/citología , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Humanos , Subtipo H1N1 del Virus de la Influenza A/fisiología , Subtipo H3N2 del Virus de la Influenza A/fisiología , Ratones , Ratones Endogámicos C57BL , Neutrófilos/citología , Neutrófilos/inmunología , Infecciones por Orthomyxoviridae/mortalidad , Infecciones por Orthomyxoviridae/patología , Péptidos/farmacología , Receptor TIE-2/metabolismo , Tasa de Supervivencia , Replicación Viral/efectos de los fármacos
16.
Anesthesiology ; 122(6): 1338-48, 2015 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-25815455

RESUMEN

BACKGROUND: Hypoxic pulmonary vasoconstriction (HPV) is critically important in regionally heterogeneous lung diseases by directing blood toward better-oxygenated lung units, yet the molecular mechanism of HPV remains unknown. Transient receptor potential (TRP) channels are a large cation channel family that has been implicated in HPV, specifically in the pulmonary artery smooth muscle cell (PASMC) Ca and contractile response to hypoxia. In this study, the authors probed the role of the TRP family member, TRPV4, in HPV. METHODS: HPV was assessed by using isolated perfused mouse lungs or by intravital microscopy to directly visualize pulmonary arterioles in mice. In vitro experiments were performed in primary human PASMC. RESULTS: The hypoxia-induced pulmonary artery pressure increase seen in wild-type mice (5.6 ± 0.6 mmHg; mean ± SEM) was attenuated both by inhibition of TRPV4 (2.8 ± 0.5 mmHg), or in lungs from TRPV4-deficient mice (Trpv4) (3.4 ± 0.5 mmHg; n = 7 each). Functionally, Trpv4 mice displayed an exaggerated hypoxemia after regional airway occlusion (paO2 71% of baseline ± 2 vs. 85 ± 2%; n = 5). Direct visualization of pulmonary arterioles by intravital microscopy revealed a 66% reduction in HPV in Trpv4 mice. In human PASMC, inhibition of TRPV4 blocked the hypoxia-induced Ca influx and myosin light chain phosphorylation. TRPV4 may form a heteromeric channel with TRPC6 as the two channels coimmunoprecipitate from PASMC and as there is no additive effect of TRPC and TRPV4 inhibition on Ca influx in response to the agonist, 11,12-epoxyeicosatrienoic acid. CONCLUSION: TRPV4 plays a critical role in HPV, potentially via cooperation with TRPC6.


Asunto(s)
Hipoxia/fisiopatología , Circulación Pulmonar/efectos de los fármacos , Receptor Toll-Like 4/efectos de los fármacos , Vasoconstricción/efectos de los fármacos , Animales , Calcio/metabolismo , Calcio/farmacología , Caveolas/efectos de los fármacos , Humanos , Técnicas In Vitro , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Músculo Liso Vascular/efectos de los fármacos , Ventilación Unipulmonar , Receptor Toll-Like 4/genética , Receptor Toll-Like 6/efectos de los fármacos , Relación Ventilacion-Perfusión
17.
Am J Respir Cell Mol Biol ; 53(4): 459-70, 2015 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-25693001

RESUMEN

A major cause of death after influenza virus infection is lung injury due to a bacterial superinfection, yet the mechanism is unknown. Death has been attributed to virus-induced immunosuppression and bacterial overgrowth, but this hypothesis is based on data from the preantibiotic era and animal models that omit antimicrobial therapy. Because of diagnostic uncertainty, most patients with influenza receive antibiotics, making bacterial overgrowth unlikely. Respiratory failure after superinfection presents as acute respiratory distress syndrome, a disorder characterized by lung microvascular leak and edema. The objective of this study was to determine whether the influenza virus sensitizes the lung endothelium to leak upon exposure to circulating bacterial-derived molecular patterns from Staphylococcus aureus. In vitro as well as in vivo models of influenza followed by S. aureus superinfection were used. Molecular mechanisms were explored using molecular biology, knockout mice, and human autopsy specimens. Influenza virus infection sensitized human lung endothelium to leak when challenged with S. aureus, even at low doses of influenza and even when the pathogens were given days apart. Influenza virus increased endothelial expression of TNFR1 both in vitro and in intact lungs, a finding corroborated by human autopsy specimens of patients with influenza. Leak was recapitulated with protein A, a TNFR1 ligand, and sequential infection caused protein A-dependent loss of IκB, cleavage of caspases 8 and 3, and lung endothelial apoptosis. Mice infected sequentially with influenza virus and S. aureus developed significantly increased lung edema that was protein A and TNFR1 dependent. Influenza virus primes the lung endothelium to leak, predisposing patients to acute respiratory distress syndrome upon exposure to S. aureus.


Asunto(s)
Endotelio Vascular/metabolismo , Gripe Humana/metabolismo , Microvasos/metabolismo , Infecciones Estafilocócicas/metabolismo , Animales , Apoptosis , Permeabilidad Capilar , Células Cultivadas , Endotelio Vascular/microbiología , Endotelio Vascular/patología , Humanos , Subtipo H3N2 del Virus de la Influenza A/fisiología , Gripe Humana/patología , Gripe Humana/virología , Pulmón/irrigación sanguínea , Ratones , Ratones Noqueados , Microvasos/microbiología , Microvasos/patología , FN-kappa B/metabolismo , Receptores de Reconocimiento de Patrones/metabolismo , Receptores Tipo I de Factores de Necrosis Tumoral/metabolismo , Infecciones Estafilocócicas/patología , Proteína Estafilocócica A/metabolismo , Staphylococcus aureus/fisiología , Regulación hacia Arriba
18.
J Biol Chem ; 290(5): 2547-59, 2015 Jan 30.
Artículo en Inglés | MEDLINE | ID: mdl-25527499

RESUMEN

Pulmonary fibrosis is a progressive disease characterized by fibroblast proliferation and excess deposition of collagen and other extracellular matrix components. Although the origin of fibroblasts is multifactorial, recent data implicate endothelial-to-mesenchymal transition as an important source of fibroblasts. We report herein that loss of the essential autophagy gene ATG7 in endothelial cells (ECs) leads to impaired autophagic flux accompanied by marked changes in EC architecture, loss of endothelial, and gain of mesenchymal markers consistent with endothelial-to-mesenchymal transition. Loss of ATG7 also up-regulates TGFß signaling and key pro-fibrotic genes in vitro. In vivo, EC-specific ATG7 knock-out mice exhibit a basal reduction in endothelial-specific markers and demonstrate an increased susceptibility to bleomycin-induced pulmonary fibrosis and collagen accumulation. Our findings help define the role of endothelial autophagy as a potential therapeutic target to limit organ fibrosis, a condition for which presently there are no effective available treatments.


Asunto(s)
Células Endoteliales/citología , Mesodermo/citología , Proteínas Asociadas a Microtúbulos/metabolismo , Enzimas Activadoras de Ubiquitina/metabolismo , Animales , Proteína 7 Relacionada con la Autofagia , Bleomicina/toxicidad , Línea Celular , Técnica del Anticuerpo Fluorescente , Células Endoteliales de la Vena Umbilical Humana/citología , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Immunoblotting , Inmunohistoquímica , Ratones , Ratones Noqueados , Proteínas Asociadas a Microtúbulos/genética , Fibrosis Pulmonar/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Enzimas Activadoras de Ubiquitina/genética
19.
PLoS One ; 8(9): e75861, 2013.
Artículo en Inglés | MEDLINE | ID: mdl-24069452

RESUMEN

RATIONALE: Stable analogs of vasoactive intestinal peptide (VIP) have been proposed as novel line of therapy in chronic obstructive pulmonary disease (COPD) based on their bronchodilatory and anti-inflammatory effects. We speculated that VIP analogs may provide additional benefits in that they exert vasodilatory properties in the lung, and tested this hypothesis in both ex vivo and in vivo models. METHODS: In isolated perfused mouse lungs and in an in vivo rat model, pulmonary blood vessels were preconstricted by hypoxia and hemodynamic changes in response to systemic (ex vivo) or inhaled (in vivo) administration of the cyclic VIP analog RO 25-1553 were determined. RESULTS: In mouse lungs, RO 25-1553 reduced intrinsic vascular resistance at normoxia, and attenuated the increase in pulmonary artery pressure in response to acute hypoxia. Consistently, inhalation of RO 25-1553 (1 mg · mL(-1) for 3 min) caused an extensive and sustained (> 60 min) inhibition of the pulmonary arterial pressure increase in response to hypoxia in vivo that was comparable to the effects of inhaled sildenafil. This effect was not attributable to systemic cardiovascular effects of RO 25-1553, but to a lung specific reduction in pulmonary vascular resistance, while cardiac output and systemic arterial hemodynamics remained unaffected. No adverse effects of RO 25-1553 inhalation on pulmonary gas exchange, ventilation-perfusion matching, or lung fluid content were detected. CONCLUSION: Our findings demonstrate that inhaled delivery of the stable VIP analog RO 25-1553 induces a potent and sustained vasodilatory effect in the pulmonary circulation with no detectable adverse effects. Therapeutic inhalation of RO 25-1553 may provide vascular benefits in addition to its reported anti-inflammatory and bronchodilatory effects in COPD, yet caution is warranted given the overall poor results of vasodilator therapies for pulmonary hypertension secondary to COPD in a series of recent clinical trials.


Asunto(s)
Pulmón/irrigación sanguínea , Pulmón/efectos de los fármacos , Péptidos Cíclicos/farmacología , Circulación Pulmonar/efectos de los fármacos , Receptores de Péptido Intestinal Vasoactivo/agonistas , Vasodilatadores/farmacología , Administración por Inhalación , Animales , Hemodinámica/efectos de los fármacos , Hipoxia , Masculino , Ratones , Péptidos Cíclicos/administración & dosificación , Péptidos Cíclicos/efectos adversos , Ratas , Péptido Intestinal Vasoactivo/administración & dosificación , Péptido Intestinal Vasoactivo/efectos adversos , Péptido Intestinal Vasoactivo/farmacología , Vasodilatadores/administración & dosificación , Vasodilatadores/efectos adversos
20.
Am J Respir Crit Care Med ; 188(4): 474-81, 2013 Aug 15.
Artículo en Inglés | MEDLINE | ID: mdl-23796161

RESUMEN

RATIONALE: Oxygen uptake is the elemental function of the lung. However, current understanding of this process has largely been derived from theoretical considerations and measurements of global pulmonary gas exchange. OBJECTIVES: To report the direct visualization of pulmonary oxygen uptake in vivo and its use for the analysis of temporal and spatial oxygenation profiles along individual arteriovenous pathways in lungs of healthy and chronic hypoxic mice. METHODS: A murine model for intravital microscopy of the breathing lung under sealed thorax conditions was combined with multispectral oximetry for two-dimensional oxygen saturation mapping. This combination allowed for visualization of the blood oxygenation process from pulmonary arterioles to capillaries and venules in two-dimensional oxygen saturation maps. MEASUREMENTS AND MAIN RESULTS: Temporal and spatial oxygenation profiles revealed that oxygenation occurs within 100 milliseconds over a distance of approximately 130 µm in the pulmonary microvasculature of the anesthetized mouse. About 50% of total oxygen uptake takes place in precapillary arterioles of less than 30 µm in diameter before the blood enters the alveolar capillary bed. In chronic hypoxic mice, precapillary oxygenation was significantly attenuated as a result of the widened transarteriolar diffusion distance. CONCLUSIONS: Oxygen saturation mapping in the intact lung yields unique insights into the temporal and spatial characteristics of pulmonary gas exchange in intact and diseased lungs. Precapillary gas exchange contributes importantly to blood oxygenation at rest, but is attenuated in remodeled lung arterioles, which may be of relevance in pulmonary hypertension.


Asunto(s)
Oxígeno/sangre , Intercambio Gaseoso Pulmonar/fisiología , Animales , Arteriolas/metabolismo , Capilares/metabolismo , Modelos Animales de Enfermedad , Hemodinámica , Hipertensión Pulmonar/fisiopatología , Hipoxia/fisiopatología , Masculino , Ratones , Ratones Endogámicos BALB C , Oximetría , Vénulas/metabolismo
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