Yuexin Shan

Neuroimmune Regulation of Ventilator-induced Lung Injury

RATIONALE Ventilator-induced lung injury (VILI) contributes to the mortality in patients with acute lung injury by increasing inflammation. Recent evidence suggests that stimulation of the cholinergic antiinflammatory pathway may be an attractive way to attenuate inflammatory injury. OBJECTIVES To determine the role of vagus nerve signaling in VILI and establish whether stimulation of the vagus reflex can mitigate VILI. METHODS We performed bilateral vagotomy in a mouse model of high-tidal volume-induced lung injury. We performed pharmacological and electrical vagus nerve stimulation in a rat model of VILI following ischemia/reperfusion injury. To determine the contribution of the alpha 7 acetylcholine nicotinic receptor to pulmonary cell injury, we exposed human bronchial epithelial cells to cyclic stretch in the presence of specific agonist or antagonist of the alpha 7 receptor. MEASUREMENTS AND MAIN RESULTS Vagotomy exacerbates lung injury from VILI in mice as demonstrated by increased wet-to-dry ratio, infiltration of neutrophils, and increased IL-6. Vagal stimulation attenuates lung injury in rats after ischemia/reperfusion injury ventilated with high-volume strategies. Treatment of both mice and rats with the vagus mimetic drug semapimod resulted in decreased lung injury. Vagotomy also increased pulmonary apoptosis, whereas vagus stimulation (electrical and pharmacological) attenuated VILI-induced apoptosis. In vitro studies suggest that vagus-dependent effects on inflammation and apoptosis are mediated via the α7 nicotinc acetylcholine receptor-dependent effects on cyclic stretch-dependent signaling pathways c-jun N-terminal kinase and tumor necrosis factor receptor superfamily, member 6. CONCLUSIONS Stimulation of the cholinergic antiinflammatory reflex may represent a promising alternative for the treatment of VILI.

Activating Transcription Factor 3 Confers Protection against Ventilator-induced Lung Injury

RATIONALE Ventilator-induced lung injury (VILI) significantly contributes to mortality in patients with acute respiratory distress syndrome, the most severe form of acute lung injury. Understanding the molecular basis for response to cyclic stretch (CS) and its derangement during high-volume ventilation is of high priority. OBJECTIVES To identify specific molecular regulators involved in the development of VILI. METHODS We undertook a comparative examination of cis-regulatory sequences involved in the coordinated expression of CS-responsive genes using microarray analysis. Analysis of stretched versus nonstretched cells identified significant enrichment for genes containing putative binding sites for the transcription factor activating transcription factor 3 (ATF3). To determine the role of ATF3 in vivo, we compared the response of ATF3 gene-deficient mice to wild-type mice in an in vivo model of VILI. MEASUREMENTS AND MAIN RESULTS ATF3 protein expression and nuclear translocation is increased in the lung after mechanical ventilation in wild-type mice. ATF3-deficient mice have greater sensitivity to mechanical ventilation alone or in conjunction with inhaled endotoxin, as demonstrated by increased cell infiltration and proinflammatory cytokines in the lung and bronchoalveolar lavage, and increased pulmonary edema and indices of tissue injury. The expression of stretch-responsive genes containing putative ATF3 cis-regulatory regions was significantly altered in ATF3-deficient mice. CONCLUSIONS ATF3 deficiency confers increased sensitivity to mechanical ventilation alone or in combination with inhaled endotoxin. We propose ATF3 acts to counterbalance CS and high volume-induced inflammation, dampening its ability to cause injury and consequently protecting animals from injurious CS.

Network Analysis of Transcriptional Responses Induced by Mesenchymal Stem Cell Treatment of Experimental Sepsis

Although bone marrow-derived mesenchymal stem cell (MSC) systemic administration reduces sepsis-associated inflammation, organ injury, and mortality in clinically relevant models of polymicrobial sepsis, the cellular and molecular mechanisms mediating beneficial effects are controversial. This study identifies the molecular mechanisms of MSC-conferred protection in sepsis by interrogating transcriptional responses of target organs to MSC therapy. Sepsis was induced in C57Bl/6J mice by cecal ligation and puncture, followed 6 hours later by an i.v. injection of either MSCs or saline. Total RNA from lungs, hearts, kidneys, livers, and spleens harvested 28 hours after cecal ligation and puncture was hybridized to mouse expression bead arrays. Common transcriptional responses were analyzed using a network knowledge-based approach. A total of 4751 genes were significantly changed between placebo- and MSC-treated mice (adjusted P ≤ 0.05). Transcriptional responses identified three common effects of MSC administration in all five organs examined: i) attenuation of sepsis-induced mitochondrial-related functional derangement, ii down-regulation of endotoxin/Toll-like receptor innate immune proinflammatory transcriptional responses, and iii) coordinated expression of transcriptional programs implicated in the preservation of endothelial/vascular integrity. Transcriptomic analysis indicates that the protective effect of MSC therapy in sepsis is not limited to a single mediator or pathway but involves a range of complementary activities affecting biological networks playing critical roles in the control of host cell metabolism and inflammatory response.

Effects of mesenchymal stem cell therapy on the time course of pulmonary remodeling depend on the etiology of lung injury in mice.

Objective:Recent evidence suggests that mesenchymal stem cells may attenuate lung inflammation and fibrosis in acute lung injury. However, so far, no study has investigated the effects of mesenchymal stem cell therapy on the time course of the structural, mechanical, and remodeling properties in pulmonary or extrapulmonary acute lung injury. Design:Prospective randomized controlled experimental study. Setting:University research laboratory. Subjects:One hundred forty-three females and 24 male C57BL/6 mice. Interventions:Control mice received saline solution intratracheally (0.05 mL, pulmonary control) or intraperitoneally (0.5 mL, extrapulmonary control). Acute lung injury mice received Escherichia coli lipopolysaccharide intratracheally (2 mg/kg in 0.05 mL of saline/mouse, pulmonary acute lung injury) or intraperitoneally (20 mg/kg in 0.5 mL of saline/mouse, extrapulmonary acute lung injury). Mesenchymal stem cells were intravenously injected (IV, 1 × 105 cells in 0.05 mL of saline/mouse) 1 day after lipopolysaccharide administration. Measurements and Main Results:At days 1, 2, and 7, static lung elastance and the amount of alveolar collapse were similar in pulmonary and extrapulmonary acute lung injury groups. Inflammation was markedly increased at day 2 in both acute lung injury groups as evidenced by neutrophil infiltration and levels of cytokines in bronchoalveolar lavage fluid and lung tissue. Conversely, collagen deposition was only documented in pulmonary acute lung injury. Mesenchymal stem cell mitigated changes in elastance, alveolar collapse, and inflammation at days 2 and 7. Compared with extrapulmonary acute lung injury, mesenchymal stem cell decreased collagen deposition only in pulmonary acute lung injury. Furthermore, mesenchymal stem cell increased metalloproteinase-8 expression and decreased expression of tissue inhibitor of metalloproteinase-1 in pulmonary acute lung injury, suggesting that mesenchymal stem cells may have an effect on the remodeling process. This change may be related to a shift in macrophage phenotype from M1 (inflammatory and antimicrobial) to M2 (wound repair and inflammation resolution) phenotype. Conclusions:Mesenchymal stem cell therapy improves lung function through modulation of the inflammatory and remodeling processes. In pulmonary acute lung injury, a reduction in collagen fiber content was observed associated with a balance between metalloproteinase-8 and tissue inhibitor of metalloproteinase-1 expressions.

Salutary effect of resveratrol on sepsis-induced myocardial depression

Objectives:We hypothesized that resveratrol administration would reverse sepsis-dependent downregulation of peroxisome proliferator activated receptor-&ggr; coactivator 1&agr;, preserve mitochondrial integrity, and rescue animals from sepsis-induced myocardial failure. Setting:Teaching hospital research laboratory. Interventions:Cecal ligation and puncture in mice was performed to induce sepsis. Mice that underwent cecal ligation and puncture were randomly assigned to receive resveratrol (30 mg/kg or 60 mg/kg) or vehicle 1 mL sodium chloride 0.9% subcutaneously in the scruff of the neck directly after surgery and at 16, 24, and 40 hrs, respectively. Measurements and Results:Forty-eight hrs after cecal ligation and puncture, cardiac performance was established using echocardiography. Mitochondrial integrity was evaluated with electron microscopy, and changes in gene expression were evaluated with microarray analysis. Survival at 48 hrs was just under 50% and comparable between groups. Myocardial contractile function significantly improved after resveratrol treatment. Resveratrol-treated mice developed focal areas of edema, whereas vehicle-treated mice developed significant, diffuse myocardial edema. Electron microscopy revealed widespread swollen mitochondria with ruptured outer membranes, autophagosomes, and vacuolation of the internal compartment, which were significantly attenuated in resveratrol-treated animals. Resveratrol treatment significantly increased cardiac expression of peroxisome proliferator–activated receptor-&ggr; coactivator 1a. Microarray analysis revealed that resveratrol treatment resulted in upregulation of the peroxisome proliferator–activated receptor-&ggr; coactivator gene set containing genes known to be regulated by this transcriptional coactivator. Our data strongly suggest that administration of resveratrol modulates bioenergy metabolism, substrate utilization, oxidative stress, and detoxification pathways associated with both mitochondrial and cardiac pathological conditions, but does not alter mortality from sepsis. Conclusions:The salutary effects of resveratrol on cecal ligation and puncture-induced myocardial dysfunction are associated with increased peroxisome proliferator–activated receptor-&ggr; coactivator 1a abundance and function. Preservation of myocardial energy production capacity, prevention of secondary injury, mitigation of inflammation, and reversal of sepsis-induced myocardial remodeling are likely to underlie its beneficial effects. This however, does not result in improved survival.

ATF3 Protects Pulmonary Resident Cells from Acute and Ventilator-Induced Lung Injury by Preventing Nrf2 Degradation

AIMS Ventilator-induced lung injury (VILI) contributes to mortality in patients with acute respiratory distress syndrome, the most severe form of acute lung injury (ALI). Absence of activating transcription factor 3 (ATF3) confers susceptibility to ALI/VILI. To identify cell-specific ATF3-dependent mechanisms of susceptibility to ALI/VILI, we generated ATF3 chimera by adoptive bone marrow (BM) transfer and randomized to inhaled saline or lipopolysacharide (LPS) in the presence of mechanical ventilation (MV). Adenovirus vectors to silence or overexpress ATF3 were used in primary human bronchial epithelial cells and murine BM-derived macrophages from wild-type or ATF3-deficient mice. RESULTS Absence of ATF3 in myeloid-derived cells caused increased pulmonary cellular infiltration. In contrast, absence of ATF3 in parenchymal cells resulted in loss of alveolar-capillary membrane integrity and increased exudative edema. ATF3-deficient macrophages were unable to limit the expression of pro-inflammatory mediators. Knockdown of ATF3 in resident cells resulted in decreased junctional protein expression and increased paracellular leak. ATF3 overexpression abrogated LPS induced membrane permeability. Despite release of ATF3-dependent Nrf2 transcriptional inhibition, mice that lacked ATF3 expression in resident cells had increased Nrf2 protein degradation. INNOVATION In our model, in the absence of ATF3 in parenchymal cells increased Nrf2 degradation is the result of increased Keap-1 expression and loss of DJ-1 (Parkinson disease [autosomal recessive, early onset] 7), previously not known to play a role in lung injury. CONCLUSION Results suggest that ATF3 confers protection to lung injury by preventing inflammatory cell recruitment and barrier disruption in a cell-specific manner, opening novel opportunities for cell specific therapy for ALI/VILI.

DJ-1/PARK7 Impairs Bacterial Clearance in Sepsis

Rationale: Effective and rapid bacterial clearance is a fundamental determinant of outcomes in sepsis. DJ‐1 is a well‐established reactive oxygen species (ROS) scavenger. Objectives: Because cellular ROS status is pivotal to inflammation and bacterial killing, we determined the role of DJ‐1 in bacterial sepsis. Methods: We used cell and murine models with gain‐ and loss‐of‐function experiments, plasma, and cells from patients with sepsis. Measurements and Main Results: Stimulation of bone marrow‐derived macrophages (BMMs) with endotoxin resulted in increased DJ‐1 mRNA and protein expression. Cellular and mitochondrial ROS was increased in DJ‐1‐deficient (−/−) BMMs compared with wild‐type. In a clinically relevant model of polymicrobial sepsis (cecal ligation and puncture), DJ‐1−/− mice had improved survival and bacterial clearance. DJ‐1−/− macrophages exhibited enhanced phagocytosis and bactericidal activity in vitro, and adoptive transfer of DJ‐1−/− bone marrow‐derived mononuclear cells rescued wild‐type mice from cecal ligation and puncture‐induced mortality. In stimulated BMMs, DJ‐1 inhibited ROS production by binding to p47phox, a critical component of the NADPH oxidase complex, disrupting the complex and facilitating Nox2 (gp91phox) ubiquitination and degradation. Knocking down DJ‐1 (siRNA) in THP‐1 (human monocytic cell line) and polymorphonuclear cells from patients with sepsis enhanced bacterial killing and respiratory burst. DJ‐1 protein levels were elevated in plasma from patients with sepsis. Higher levels of circulating DJ‐1 were associated with increased organ failure and death. Conclusions: These novel findings reveal DJ‐1 impairs optimal ROS production for bacterial killing with important implications for host survival in sepsis.

Systemic angiopoietin-1/2 dysregulation following cardiopulmonary bypass in adults

Aim: Vascular leakage following cardiopulmonary bypass contributes to morbidity. Angiopoietin-1 and -2 are biomarkers of endothelial dysfunction. Our aim was to characterize Ang-1 and -2 association with clinical characteristics and outcomes. Methods: Observational cohort study measuring Ang-1/-2 with a panel of cytokines in adults undergoing cardiopulmonary bypass. Results: Ang-2 levels increased immediately postop whereas Ang-1 levels decreased over time. No significant correlation was found with other inflammatory mediators. High correlation was found between the hospital length of stay and Ang-2 increase at 24 h (rho = 0.590; p < 0.0001). The predictors of Ang-2 increase were female gender, cross clamp time, transfusion of blood and absence of angiotensin-converting enzyme inhibitor as a pre-op medication. Conclusion: Angiopoietins can detect vascular leakage early and could impact patients management to decrease length of stay after cardiac surgery.

0450. Systemic dysregulation of the angiopoietin-1/2 system in adults undergoing cardiopulmonary bypass (CBP)

Systemic capillary leak syndrome after cardiopulmonary bypass (CBP) is a well-known phenomenon, accompanied by interstitial fluid accumulation and inflammation, and can lead to end-organ failure or increased hospital length of stay (LOS). The pathophysiology responsible for capillary leak involves activation of inflammation, complement, and coagulation. The endothelium plays a central role in the activation of coagulation, and activated/dysregulated endothelium contributes to microvascular leak and enhanced adhesion of leukocytes. However, the characterization and measurement of endothelial dysregulation is not well established. Angiopoietin-1 (Ang-1) and -2 (Ang-2) are molecules implicated in angiogenesis and regulation of endothelial permeability. Their differential changes of expression could be a surrogate to quantify endothelial dysregulation after CBP.