Endothelial Heterogeneity in the Response to Autophagy Drives Small Vessel Muscularization in Pulmonary Hypertension.

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.

Platelet extracellular vesicles mediate transfusion-related acute lung injury by imbalancing the sphingolipid rheostat.

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.

Cigarette smoke augments CSF3 expression in neutrophils to compromise alveolar-capillary barrier function during influenza infection.

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.

Pulsatility damping in the microcirculation: Basic pattern and modulating factors.

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.

T regulatory cells and dendritic cells protect against transfusion-related acute lung injury via IL-10.

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.

The mast cell-B cell axis in lung vascular remodeling and pulmonary hypertension.

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.

Acid sphingomyelinase mediates murine acute lung injury following transfusion of aged platelets.

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.

Acute Lung Injury Causes Asynchronous Alveolar Ventilation That Can Be Corrected by Individual Sighs.

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.

Role of Transient Receptor Potential Vanilloid 4 in Neutrophil Activation and Acute Lung Injury.

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.

The essential autophagy gene ATG7 modulates organ fibrosis via regulation of endothelial-to-mesenchymal transition.

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.

Chloride transport-driven alveolar fluid secretion is a major contributor to cardiogenic lung edema.

Alveolar fluid clearance driven by active epithelial Na(+) and secondary Cl(-) absorption counteracts edema formation in the intact lung. Recently, we showed that impairment of alveolar fluid clearance because of inhibition of epithelial Na(+) channels (ENaCs) promotes cardiogenic lung edema. Concomitantly, we observed a reversal of alveolar fluid clearance, suggesting that reversed transepithelial ion transport may promote lung edema by driving active alveolar fluid secretion. We, therefore, hypothesized that alveolar ion and fluid secretion may constitute a pathomechanism in lung edema and aimed to identify underlying molecular pathways. In isolated perfused lungs, alveolar fluid clearance and secretion were determined by a double-indicator dilution technique. Transepithelial Cl(-) secretion and alveolar Cl(-) influx were quantified by radionuclide tracing and alveolar Cl(-) imaging, respectively. Elevated hydrostatic pressure induced ouabain-sensitive alveolar fluid secretion that coincided with transepithelial Cl(-) secretion and alveolar Cl(-) influx. Inhibition of either cystic fibrosis transmembrane conductance regulator (CFTR) or Na(+)-K(+)-Cl(-) cotransporters (NKCC) blocked alveolar fluid secretion, and lungs of CFTR(-/-) mice were protected from hydrostatic edema. Inhibition of ENaC by amiloride reproduced alveolar fluid and Cl(-) secretion that were again CFTR-, NKCC-, and Na(+)-K(+)-ATPase-dependent. Our findings show a reversal of transepithelial Cl(-) and fluid flux from absorptive to secretory mode at hydrostatic stress. Alveolar Cl(-) and fluid secretion are triggered by ENaC inhibition and mediated by NKCC and CFTR. Our results characterize an innovative mechanism of cardiogenic edema formation and identify NKCC1 as a unique therapeutic target in cardiogenic lung edema.

Influenza-Induced Priming and Leak of Human Lung Microvascular Endothelium upon Exposure to Staphylococcus aureus.

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.

TRPV4 Is Required for Hypoxic Pulmonary Vasoconstriction.

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.

Precapillary oxygenation contributes relevantly to gas exchange in the intact lung.

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.

Microparticles and acute lung injury.

The pathophysiology of acute lung injury (ALI) and its most severe form, acute respiratory distress syndrome (ARDS), is characterized by increased vascular and epithelial permeability, hypercoagulation and hypofibrinolysis, inflammation, and immune modulation. These detrimental changes are orchestrated by cross talk between a complex network of cells, mediators, and signaling pathways. A rapidly growing number of studies have reported the appearance of distinct populations of microparticles (MPs) in both the vascular and alveolar compartments in animal models of ALI/ARDS or respective patient populations, where they may serve as diagnostic and prognostic biomarkers. MPs are small cytosolic vesicles with an intact lipid bilayer that can be released by a variety of vascular, parenchymal, or blood cells and that contain membrane and cytosolic proteins, organelles, lipids, and RNA supplied from and characteristic for their respective parental cells. Owing to this endowment, MPs can effectively interact with other cell types via fusion, receptor-mediated interaction, uptake, or mediator release, thereby acting as intrinsic stimulators, modulators, or even attenuators in a variety of disease processes. This review summarizes current knowledge on the formation and potential functional role of different MPs in inflammatory diseases with a specific focus on ALI/ARDS. ALI has been associated with the formation of MPs from such diverse cellular origins as platelets, neutrophils, monocytes, lymphocytes, red blood cells, and endothelial and epithelial cells. Because of their considerable heterogeneity in terms of origin and functional properties, MPs may contribute via both harmful and beneficial effects to the characteristic pathological features of ALI/ARDS. A better understanding of the formation, function, and relevance of MPs may give rise to new promising therapeutic strategies to modulate coagulation, inflammation, endothelial function, and permeability either through removal or inhibition of "detrimental" MPs or through administration or stimulation of "favorable" MPs.

Recipient T lymphocytes modulate the severity of antibody-mediated transfusion-related acute lung injury.

Transfusion-related acute lung injury (TRALI) is a serious complication of transfusion and has been ranked as one of the leading causes of transfusion-related fatalities. Nonetheless, many details of the immunopathogenesis of TRALI, particularly with respect to recipient factors are unknown. We used a murine model of antibody-mediated TRALI in an attempt to understand the role that recipient lymphocytes might play in TRALI reactions. Intravenous injection of an IgG2a antimurine major histocompatibility complex class I antibody (34-1-2s) into BALB/c mice induced moderate hypothermia and pulmonary granulocyte accumulation but no pulmonary edema nor mortality. In contrast, 34-1-2s injections into mice with severe combined immunodeficiency caused severe hypothermia, severe pulmonary edema, and approximately 40% mortality indicating a critical role for T and B lymphocytes in suppressing TRALI reactions. Adoptive transfer of purified CD8(+) T lymphocytes or CD4(+) T cells but not CD19(+) B cells into the severe combined immunodeficiency mice alleviated the antibody-induced hypothermia, lung damage, and mortality, suggesting that T lymphocytes were responsible for the protective effect. Taken together, these results suggest that recipient T lymphocytes play a significant role in suppressing antibody-mediated TRALI reactions. They identify a potentially new recipient mechanism that controls the severity of TRALI reactions.

Human neutrophil peptides mediate endothelial-monocyte interaction, foam cell formation, and platelet activation.

OBJECTIVE: Neutrophils are involved in the inflammatory responses during atherosclerosis. Human neutrophil peptides (HNPs) released from activated neutrophils exert immune modulating properties. We hypothesized that HNPs play an important role in neutrophil-mediated inflammatory cardiovascular responses in atherosclerosis. METHODS AND RESULTS: We examined the role of HNPs in endothelial-leukocyte interaction, platelet activation, and foam cell formation in vitro and in vivo. We demonstrated that stimulation of human coronary artery endothelial cells with clinically relevant concentrations of HNPs resulted in monocyte adhesion and transmigration; induction of oxidative stress in human macrophages, which accelerates foam cell formation; and activation and aggregation of human platelets. The administration of superoxide dismutase or anti-CD36 antibody reduced foam cell formation and cholesterol efflux. Mice deficient in double genes of low-density lipoprotein receptor and low-density lipoprotein receptor-related protein (LRP), and mice deficient in a single gene of LRP8, the only LRP phenotype expressed in platelets, showed reduced leukocyte rolling and decreased platelet aggregation and thrombus formation in response to HNP stimulation. CONCLUSIONS: HNPs exert proatherosclerotic properties that appear to be mediated through LRP8 signaling pathways, suggesting an important role for HNPs in the development of inflammatory cardiovascular diseases.

Intra-vital imaging of mesenchymal stromal cell kinetics in the pulmonary vasculature during infection.

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.

Characterization of the antiplatelet effect of aspirin at enrollment and after 2-year follow-up in a real clinical setting in Japan.

BACKGROUND: Aspirin is an antiplatelet drug widely used for the prevention of cardiovascular diseases. It has been reported that some patients who exhibit a reduced antiplatelet effect of aspirin have higher cardiovascular risk. It is still controversial whether the antiplatelet effect of aspirin diminishes after a few years of treatment. This study aimed to evaluate the antiplatelet effect of aspirin and its 2-year change in Japanese patients. METHODS AND RESULTS: Collagen-induced platelet-aggregability was measured at enrollment by conventional optical aggregometer in 239 patients undergoing antiplatelet therapy with aspirin alone. Among them, 167 patients were evaluated after 2 years. Whole blood aggregability based on the screen-filtration method was also evaluated. Optical aggregometer studies showed that 27% of patients were low-responders. Multivariate analyses revealed that female sex and non-use of calcium-channel blockers were associated with low responsiveness. The antiplatelet effect of aspirin did not decrease after 2 years. Similar data were obtained with the whole blood aggregometer. CONCLUSIONS: In this Japanese patient group, 27% were low-responders to aspirin, and the antiplatelet effect of aspirin did not decrease after a 2-year interval.

Ventilation and Perfusion at the Alveolar Level: Insights From Lung Intravital Microscopy.

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.