Brijesh V. Patel

TNF-Induced Death Signaling Triggers Alveolar Epithelial Dysfunction in Acute Lung Injury

The ability of the alveolar epithelium to prevent and resolve pulmonary edema is a crucial determinant of morbidity and mortality in acute lung injury (ALI). TNF has been implicated in ALI pathogenesis, but the precise mechanisms remain undetermined. We evaluated the role of TNF signaling in pulmonary edema formation in a clinically relevant mouse model of ALI induced by acid aspiration and investigated the effects of TNF p55 receptor deletion, caspase-8 inhibition, and alveolar macrophage depletion on alveolar epithelial function. We found that TNF plays a central role in the development of pulmonary edema in ALI through activation of p55-mediated death signaling, rather than through previously well-characterized p55-mediated proinflammatory signaling. Acid aspiration produced pulmonary edema with significant alveolar epithelial dysfunction, as determined by alveolar fluid clearance (AFC) and intra-alveolar levels of the receptor for advanced glycation end-products. The impairment of AFC was strongly correlated with lung caspase-8 activation, which was localized to type 1 alveolar epithelial cells by flow cytometric analysis. p55-deficient mice displayed markedly attenuated injury, with improved AFC and reduced caspase-8 activity but no differences in downstream cytokine/chemokine production and neutrophil recruitment. Caspase-8 inhibition significantly improved AFC and oxygenation, whereas depletion of alveolar macrophages attenuated epithelial dysfunction with reduced TNF production and caspase-8 activity. These results provide in vivo evidence for a novel role for TNF p55 receptor–mediated caspase-8 signaling, without substantial apoptotic cell death, in triggering alveolar epithelial dysfunction and determining the early pathophysiology of ALI. Blockade of TNF-induced death signaling may provide an effective early-phase strategy for ALI.

Resolution of acute lung injury and inflammation: a translational mouse model

Previous animal models of acute lung injury (ALI) are limited as they only reproduce part of the complex pathobiology of clinical ALI. Here we develop a translational mouse model of ALI, which not only reflects the major clinical and pathological features but also enables investigation into ALI resolution. Anaesthetised mice underwent orotracheal instillation of hydrochloric acid. During the immediate period after instillation, mice were carefully maintained with supplemental oxygen to avoid mortality. At specified time-points, lung injury was assessed by analysis of blood gases, respiratory mechanics, bronchoalveolar lavage fluid, alveolar fluid clearance and lung histology. Animals exhibited significant weight loss, decreased oxygenation, increased respiratory elastance and pulmonary inflammation (intra-alveolar leukocyte influx/cytokine levels and histological injury scores). Moreover, mice displayed alveolar–capillary barrier dysfunction/epithelial injury as reflected by increased alveolar protein, lung wet/dry weight ratio and soluble receptor for advanced glycation end-products, as well as reduced alveolar fluid clearance. These injury parameters peaked between days 1 and 3, followed by almost complete recovery over days 5–10. Histology showed evidence of fibrosis on day 10. The results indicate that this resolving model of acid aspiration represents a powerful experimental tool to investigate the injurious, inflammatory, fibrotic, and resolving and reparative processes of ALI.

Ventilation with "clinically relevant" high tidal volumes does not promote stretch-induced injury in the lungs of healthy mice

Objective: Ventilator-induced lung injury is a crucial determinant of the outcome of mechanically ventilated patients. Increasing numbers of mouse studies have identified numerous pathways and mediators that are modulated by ventilation, but it is conceptually difficult to reconcile these into a single paradigm. There is substantial variability in tidal volumes used in these studies and no certainty about the pathophysiology that such varied models actually represent. This study was designed to investigate whether ventilation strategies ranging from “very high” to more “clinically relevant” tidal volumes induce similar pathophysiologies in healthy mice or represent distinct entities. Design: In vivo study. Setting: University research laboratory. Subjects: C57/Bl6 mice. Interventions: Anesthetized mice were ventilated with various tidal volumes up to 40 mL/kg. Measurements and Main Results: Respiratory system compliance and arterial blood gases were used to evaluate physiological variables of injury. Lung wet:dry weight ratio, lavage fluid protein, and cytokines were used to assess pulmonary edema and inflammation. All ventilation strategies induced changes in respiratory system compliance, although the pattern of change was unique for each strategy. Ventilation with 10 mL/kg and 40 mL/kg also induced decreases in arterial PO2 and blood pressure. Any physiological changes induced during the 10, 20, and 30 mL/kg strategies were largely reversed by recruitment maneuvers at the end of the protocol. Markers of pulmonary edema and inflammation indicated that only 40 mL/kg induced substantial increases in both, consistent with development of lung injury. Conclusions: Tidal volumes up to 20 mL/kg are unlikely to induce substantial lung overstretch in models using healthy, young mice. Signs of injury/inflammation using such models are likely to result from other factors, particularly alveolar derecruitment and atelectasis. The results of such studies may need to be reevaluated before clinical relevance can be accurately determined.

In vivo compartmental analysis of leukocytes in mouse lungs

The lung has a unique structure consisting of three functionally different compartments (alveolar, interstitial, and vascular) situated in an extreme proximity. Current methods to localize lung leukocytes using bronchoalveolar lavage and/or lung perfusion have significant limitations for determination of location and phenotype of leukocytes. Here we present a novel method using in vivo antibody labeling to enable accurate compartmental localization/quantification and phenotyping of mouse lung leukocytes. Anesthetized C57BL/6 mice received combined in vivo intravenous and intratracheal labeling with fluorophore-conjugated anti-CD45 antibodies, and lung single-cell suspensions were analyzed by flow cytometry. The combined in vivo intravenous and intratracheal CD45 labeling enabled robust separation of the alveolar, interstitial, and vascular compartments of the lung. In naive mice, the alveolar compartment consisted predominantly of resident alveolar macrophages. The interstitial compartment, gated by events negative for both intratracheal and intravenous CD45 staining, showed two conventional dendritic cell populations, as well as a Ly6Clo monocyte population. Expression levels of MHCII on these interstitial monocytes were much higher than on the vascular Ly6Clo monocyte populations. In mice exposed to acid aspiration-induced lung injury, this protocol also clearly distinguished the three lung compartments showing the dynamic trafficking of neutrophils and exudative monocytes across the lung compartments during inflammation and resolution. This simple in vivo dual-labeling technique substantially increases the accuracy and depth of lung flow cytometric analysis, facilitates a more comprehensive examination of lung leukocyte pools, and enables the investigation of previously poorly defined “interstitial” leukocyte populations during models of inflammatory lung diseases.

Sources of alveolar soluble TNF receptors during acute lung injury of different etiologies

Elevated soluble tumor necrosis factor-α receptor (sTNFR) levels in bronchoalveolar lavage fluid (BALF) are associated with poor patient outcome in acute lung injury (ALI). The mechanisms underlying these increases are unknown, but it is possible that pulmonary inflammation and increased alveolar epithelial permeability may individually contribute. We investigated mechanisms of elevated BALF sTNFRs in two in vivo mouse models of ALI. Anesthetized mice were challenged with intratracheal lipopolysaccharide or subjected to injurious mechanical ventilation. Lipopolysaccharide instillation produced acute intra-alveolar inflammation, but minimal alveolar epithelial permeability changes, with increased BALF sTNFR p75, but not p55. Increased p75 levels were markedly attenuated by alveolar macrophage depletion. In contrast, injurious ventilation induced substantial alveolar epithelial permeability, with increased BALF p75 and p55, which strongly correlated with total protein. BALF sTNFRs were not increased in isolated buffer-perfused lungs (devoid of circulating sTNFRs) subjected to injurious ventilation. These results suggest that lipopolysaccharide-induced intra-alveolar inflammation upregulates alveolar macrophage-mediated production of sTNFR p75, whereas enhanced alveolar epithelial permeability following mechanical ventilation leads to increased BALF p75 and p55 via plasma leakage. These data provide new insights into differential regulation of intra-alveolar sTNFR levels during ALI and may suggest sTNFRs as potential markers for evaluating the pathophysiology of ALI.

Inhibition of TNF Receptor p55 By a Domain Antibody Attenuates the Initial Phase of Acid-Induced Lung Injury in Mice

Background Tumor necrosis factor-α (TNF) is strongly implicated in the development of acute respiratory distress syndrome (ARDS), but its potential as a therapeutic target has been hampered by its complex biology. TNF signals through two receptors, p55 and p75, which play differential roles in pulmonary edema formation during ARDS. We have recently shown that inhibition of p55 by a novel domain antibody (dAb™) attenuated ventilator-induced lung injury. In the current study, we explored the efficacy of this antibody in mouse models of acid-induced lung injury to investigate the longer consequences of treatment. Methods We employed two acid-induced injury models, an acute ventilated model and a resolving spontaneously breathing model. C57BL/6 mice were pretreated intratracheally or intranasally with p55-targeting dAb or non-targeting “dummy” dAb, 1 or 4 h before acid instillation. Results Acid instillation in the dummy dAb group caused hypoxemia, increased respiratory system elastance, pulmonary inflammation, and edema in both the ventilated and resolving models. Pretreatment with p55-targeting dAb significantly attenuated physiological markers of ARDS in both models. p55-targeting dAb also attenuated pulmonary inflammation in the ventilated model, with signs that altered cytokine production and leukocyte recruitment persisted beyond the very acute phase. Conclusion These results demonstrate that the p55-targeting dAb attenuates lung injury and edema formation in models of ARDS induced by acid aspiration, with protection from a single dose lasting up to 24 h. Together with our previous data, the current study lends support toward the clinical targeting of p55 for patients with, or at risk of ARDS.

Extracorporeal Circulatory Support in Acute Coronary Syndromes: A Systematic Review and Meta-Analysis

Objectives: Extracorporeal circulatory support is a life-saving technique, and its use is increasing in acute coronary syndromes. A meta-analysis on pooled event rate of short-term mortality and complications of acute coronary syndrome patients treated with extracorporeal circulatory support was performed. Data Sources: Articles were searched in MEDLINE, Cochrane Library, Google Scholar, and Biomed Central. Study Selection: Inclusion criteria were observational studies on acute coronary syndrome patients treated with extracorporeal circulatory support. Primary outcome was short-term mortality. Secondary outcomes were extracorporeal circulatory support–related complications, causes of death, long-term mortality, and bridge therapy. Data Extraction: Sixteen articles were selected. Data about clinical characteristics, acute coronary syndrome diagnosis and treatment, extracorporeal circulatory support setting, outcome definitions, and event rate were retrieved from the articles. Random effect meta-analytic pooling was performed reporting results as a summary point estimate and 95% CI. Data Synthesis: A total of 739 patients were included (mean age, 59.8 ± 2.9). The event rate of short-term mortality was 58% (95% CI, 51–64%), 6-month mortality was affecting 24% (95% CI, 5–63%) of 1-month survivors, and 1-year mortality 17% (95% CI, 6–40%) of 6-month survivors. The event rates of extracorporeal circulatory support–related complications were acute renal failure 41%, bleeding 25%, neurologic damage in survivors 21%, sepsis/infections 21%, and leg ischemia 12%. Between causes of death, multiple organ failure and brain death affected respectively 40% and 27% of patients. Bridge to ventricular assistance device was offered to 14% of patients, and 7% received a transplant. Conclusions: There is still a high rate of short-term mortality and complications in acute coronary syndrome patients treated with extracorporeal circulatory support. New studies are needed to optimize and standardize extracorporeal circulatory support.

New Consensus Definitions for Sepsis and Septic Shock: Implications for Treatment Strategies and Drug Development?

Sepsis continues to escape a precise diagnostic definition. The most recent consensus definition, termed Sepsis-3, highlights the importance of the maladaptive and potentially life-threatening host response to infection. After briefly reviewing the history and epidemiology of sepsis, we go on to describe some of the challenges encountered when classifying such a heterogenous disease state. In the context of these new definitions for sepsis and septic shock, we explore current and potentially novel therapies, and conclude by mentioning some of the controversies of this most recent framework.

High fat feeding protects mice from ventilator-induced lung injury, via neutrophil-independent mechanisms

Objective: Obesity has a complex impact on acute respiratory distress syndrome patients, being associated with increased likelihood of developing the syndrome but reduced likelihood of dying. We propose that such observations are potentially explained by a model in which obesity influences the iatrogenic injury that occurs subsequent to intensive care admission. This study therefore investigated whether fat feeding protected mice from ventilator-induced lung injury. Design: In vivo study. Setting: University research laboratory. Subjects: Wild-type C57Bl/6 mice or tumor necrosis factor receptor 2 knockout mice, either fed a high-fat diet for 12–14 weeks, or age-matched lean controls. Interventions: Anesthetized mice were ventilated with injurious high tidal volume ventilation for periods up to 180 minutes. Measurements and Main Results: Fat-fed mice showed clear attenuation of ventilator-induced lung injury in terms of respiratory mechanics, blood gases, and pulmonary edema. Leukocyte recruitment and activation within the lungs were not significantly attenuated nor were a host of circulating or intra-alveolar inflammatory cytokines. However, intra-alveolar matrix metalloproteinase activity and levels of the matrix metalloproteinase cleavage product soluble receptor for advanced glycation end products were significantly attenuated in fat-fed mice. This was associated with reduced stretch-induced CD147 expression on lung epithelial cells. Conclusions: Consumption of a high-fat diet protects mice from ventilator-induced lung injury in a manner independent of neutrophil recruitment, which we postulate instead arises through blunted up-regulation of CD147 expression and subsequent activation of intra-alveolar matrix metalloproteinases. These findings may open avenues for therapeutic manipulation in acute respiratory distress syndrome and could have implications for understanding the pathogenesis of lung disease in obese patients.

Unloading of Right Ventricle and Clinical Improvement after Ultrasound-Accelerated Thrombolysis in Patients with Submassive Pulmonary Embolism

Acute pulmonary embolism (PE) can be devastating. It is classified into three categories based on clinical scenario, elevated biomarkers, radiographic or echocardiographic features of right ventricular strain, and hemodynamic instability. Submassive PE is diagnosed when a patient has elevated biomarkers, CT-scan, or echocardiogram showing right ventricular strain and no signs of hemodynamic compromise. Thromboemboli in the acute setting increase pulmonary vascular resistance by obstruction and vasoconstriction, resulting in pulmonary hypertension. This, further, deteriorates symptoms and hemodynamic status. Studies have shown that elevated biomarkers and right ventricular (RV) dysfunction have been associated with increased risk of mortality. Therefore, aggressive treatment is necessary to “unload” right ventricle. The treatment of submassive PE with thrombolysis is controversial, though recent data have favored thrombolysis over conventional anticoagulants in acute setting. The most feared complication of systemic thrombolysis is intracranial or major bleeding. To circumvent this problem, a newer and safer approach is sought. Ultrasound-accelerated thrombolysis is a relatively newer and safer approach that requires local administration of thrombolytic agents. Herein, we report a case series of five patients who underwent ultrasound-accelerated thrombolysis with notable improvement in symptoms and right ventricular function.