Peter Rosenberger

Hypoxia-inducible factor–dependent induction of netrin-1 dampens inflammation caused by hypoxia

The neuronal guidance molecule netrin-1 is linked to the coordination of inflammatory responses. Given that mucosal surfaces are particularly prone to hypoxia-elicited inflammation, we sought to determine the function of netrin-1 in hypoxia-induced inflammation. We detected hypoxia-inducible factor 1α (HIF-1α)-dependent induction of expression of the gene encoding netrin-1 (Ntn1) in hypoxic epithelia. Neutrophil transepithelial migration studies showed that by engaging A2B adenosine receptor (A2BAR) on neutrophils, netrin-1 attenuated neutrophil transmigration. Exogenous netrin-1 suppressed hypoxia-elicited inflammation in wild-type but not in A2BAR-deficient mice, and inflammatory hypoxia was enhanced in Ntn1+/− mice relative to that in Ntn1+/+ mice. Our studies demonstrate that HIF-1α-dependent induction of netrin-1 attenuates hypoxia-elicited inflammation at mucosal surfaces.

Identification of Ectonucleotidases CD39 and CD73 in Innate Protection during Acute Lung Injury

Acute lung injury (ALI), such as that which occurs with mechanical ventilation, contributes to morbidity and mortality of critical illness. Nonetheless, in many instances, ALI resolves spontaneously through unknown mechanisms. Therefore, we hypothesized the presence of innate adaptive pathways to protect the lungs during mechanical ventilation. In this study, we used ventilator-induced lung injury as a model to identify endogenous mechanisms of lung protection. Initial in vitro studies revealed that supernatants from stretch-induced injury contained a stable factor which diminished endothelial leakage. This factor was subsequently identified as adenosine. Additional studies in vivo revealed prominent increases in pulmonary adenosine levels with mechanical ventilation. Because ectoapyrase (CD39) and ecto-5′-nucleotidase (CD73) are rate limiting for extracellular adenosine generation, we examined their contribution to ALI. In fact, both pulmonary CD39 and CD73 are induced by mechanical ventilation. Moreover, we observed pressure- and time-dependent increases in pulmonary edema and inflammation in ventilated cd39−/− mice. Similarly, pharmacological inhibition or targeted gene deletion of cd73 was associated with increased symptom severity of ventilator-induced ALI. Reconstitution of cd39−/− or cd73−/− mice with soluble apyrase or 5′-nucleotidase, respectively, reversed such increases. In addition, ALI was significantly attenuated and survival improved after i.p. treatment of wild-type mice with soluble apyrase or 5′-nucleotidase. Taken together, these data reveal a previously unrecognized role for CD39 and CD73 in lung protection and suggest treatment with their soluble compounds as a therapeutic strategy for noninfectious ALI.

Effect of Remote Ischemic Preconditioning on Kidney Injury Among High-Risk Patients Undergoing Cardiac Surgery: A Randomized Clinical Trial

IMPORTANCE No interventions have yet been identified to reduce the risk of acute kidney injury in the setting of cardiac surgery. OBJECTIVE To determine whether remote ischemic preconditioning reduces the rate and severity of acute kidney injury in patients undergoing cardiac surgery. DESIGN, SETTING, AND PARTICIPANTS In this multicenter trial, we enrolled 240 patients at high risk for acute kidney injury, as identified by a Cleveland Clinic Foundation score of 6 or higher, between August 2013 and June 2014 at 4 hospitals in Germany. We randomized them to receive remote ischemic preconditioning or sham remote ischemic preconditioning (control). All patients completed follow-up 30 days after surgery and were analyzed according to the intention-to-treat principle. INTERVENTIONS Patients received either remote ischemic preconditioning (3 cycles of 5-minute ischemia and 5-minute reperfusion in one upper arm after induction of anesthesia) or sham remote ischemic preconditioning (control), both via blood pressure cuff inflation. MAIN OUTCOMES AND MEASURES The primary end point was the rate of acute kidney injury defined by Kidney Disease: Improving Global Outcomes criteria within the first 72 hours after cardiac surgery. Secondary end points included use of renal replacement therapy, duration of intensive care unit stay, occurrence of myocardial infarction and stroke, in-hospital and 30-day mortality, and change in acute kidney injury biomarkers. RESULTS Acute kidney injury was significantly reduced with remote ischemic preconditioning (45 of 120 patients [37.5%]) compared with control (63 of 120 patients [52.5%]; absolute risk reduction, 15%; 95% CI, 2.56%-27.44%; P = .02). Fewer patients receiving remote ischemic preconditioning received renal replacement therapy (7 [5.8%] vs 19 [15.8%]; absolute risk reduction, 10%; 95% CI, 2.25%-17.75%; P = .01), and remote ischemic preconditioning reduced intensive care unit stay (3 days [interquartile range, 2-5]) vs 4 days (interquartile range, 2-7) (P = .04). There was no significant effect of remote ischemic preconditioning on myocardial infarction, stroke, or mortality. Remote ischemic preconditioning significantly attenuated the release of urinary insulinlike growth factor-binding protein 7 and tissue inhibitor of metalloproteinases 2 after surgery (remote ischemic preconditioning, 0.36 vs control, 0.97 ng/mL2/1000; difference, 0.61; 95% CI, 0.27-0.86; P < .001). No adverse events were reported with remote ischemic preconditioning. CONCLUSIONS AND RELEVANCE Among high-risk patients undergoing cardiac surgery, remote ischemic preconditioning compared with no ischemic preconditioning significantly reduced the rate of acute kidney injury and use of renal replacement therapy. The observed reduction in the rate of acute kidney injury and the need for renal replacement warrants further investigation. TRIAL REGISTRATION German Clinical Trials Register Identifier: DRKS00005333.

Hypoxia Inducible Factor (HIF)-1 Coordinates Induction of Toll-Like Receptors TLR2 and TLR6 during Hypoxia

Background During acute infection and inflammation, dramatic shifts in tissue metabolism are typical, thereby resulting in profound tissue hypoxia. Therefore, we pursued the hypothesis, that tissue hypoxia may influence innate immune responses by transcriptional modulation of Toll-like receptor (TLRs) expression and function. Methodology/Principal Findings We gained first insight from transcriptional profiling of murine dendritic cells exposed to hypoxia (2% oxygen for 24 h). While transcript levels of other TLRs remained unchanged, we found a robust induction of TLR2 (2.36±0.7-fold; P<0.05) and TLR6 (3.46±1.56-fold; P<0.05). Additional studies in different cells types and cell-lines including human dendritic cells, monocytic cells (MM6), endothelia (HMEC-1) or intestinal epithelia (Caco-2) confirmed TLR2 and TLR6 induction of transcript, protein and function during hypoxia. Furthermore, analysis of the putative TLR2 and TLR6 promoters revealed previously unrecognized binding sites for HIF-1, which were shown by chromatin immunoprecipitation to bind the pivotal hypoxia-regulating transcription factor HIF-1alpha. Studies using loss and gain of function of HIF-1 confirmed a critical role of HIF-1alpha in coordinating TLR2 and TLR6 induction. Moreover, studies of murine hypoxia (8% oxygen over 6 h) showed TLR2 and TLR 6 induction in mucosal organs in vivo. In contrast, hypoxia induction of TLR2 and TLR6 was abolished in conditional HIF-1α mutant mice. Conclusions/Significance Taking together, these studies reveal coordinated induction of TLR2 and TLR6 during hypoxia and suggest tissue hypoxia in transcriptional adaptation of innate immune responses during acute infection or inflammation.

Hypoxia-inducible factor-dependent repression of equilibrative nucleoside transporter 2 attenuates mucosal inflammation during intestinal hypoxia.

BACKGROUND & AIMS The surface of the intestinal mucosa is particularly prone to hypoxia-induced inflammation. Previous studies implicated signaling via extracellular adenosine in endogenous attenuation of intestinal inflammation; we investigated whether epithelial adenosine transport could reduce hypoxia-induced inflammation of the mucosa. METHODS We performed in vitro studies of epithelial adenosine uptake and nucleoside transport using cultured epithelial cells. In vivo studies of ambient hypoxia levels were performed using mice with conditional loss of hypoxia-inducible factor (HIF)-alpha expression in the colon. RESULTS Studies of epithelial adenosine transport under hypoxic conditions showed that extracellular adenosine uptake occurs mainly at the apical surface of epithelial cells and is attenuated by hypoxia. Subsequent transcriptional studies suggested high expression levels of the equilibrative nucleoside transporter-2 (ENT2) in human epithelial cells and revealed ENT2 repression during hypoxia. Studies with promoter constructs, including site-directed mutagenesis, transcription factor binding assays, and HIF loss and gain of function showed a central role of HIF-1alpha in transcriptional repression of ENT2 during hypoxia. Similarly, transcriptional repression of ENT2 by ambient hypoxia was abolished in conditional HIF-1alpha mutant mice in vivo. Functional studies using RNA interference showed that loss of epithelial ENT2 was associated with reduced adenosine uptake in vitro, whereas pharmacologic inhibition of ENT2 attenuated hypoxia-induced inflammation of the mucosa in vivo. CONCLUSIONS HIF-1alpha-dependent repression of ENT2 increases mucosal adenosine signaling and attenuates hypoxia-associated inflammation of the intestine.

The Usefulness of Transesophageal Echocardiography During Intraoperative Cardiac Arrest in Noncardiac Surgery

According to guidelines established by the American Society of Anesthesiologists and the Society of Cardiovascular Anesthesiologists, life-threatening hemodynamic disturbances are classified as a category I indication for the intraoperative use of transesophageal echocardiography (TEE). However, the usefulness of TEE during intraoperative cardiac arrest and its impact on patient management have not been rigorously investigated. Using our departmental TEE database, we identified a population of 22 patients who underwent noncardiac surgical procedures and experienced unexpected intraoperative hemodynamic collapse requiring the initiation of Advanced Cardiac Life Support procedures between the time of induction of general anesthesia and the termination of the surgical procedure. Results of TEE examinations, patient records, detailed operative records, and outcome of patients were reviewed for the utility of TEE to diagnose the etiology of the hemodynamic collapse. Furthermore, the impact on subsequent patient management was evaluated. A primary suspected diagnosis of the underlying pathological process was established in 19 of 22 patients with TEE, including 9 with thromboembolic events, 6 with acute myocardial ischemia, 2 with hypovolemia, and 2 patients with pericardial tamponade. A definitive diagnosis could not be made in 3 patients with TEE. In 18 patients, TEE guided specific management beyond implementation of Advanced Cardiac Life Support protocols, including the addition of surgical procedures in 12 patients. Fourteen patients survived to leave the operating room, and 7 of these patients were eventually discharged from the hospital. Thus, TEE may provide additional diagnostic information in patients with intraoperative cardiac arrest and may directly guide specific, potentially life-saving therapy.

Netrin-1 Dampens Pulmonary Inflammation during Acute Lung Injury

RATIONALE Acute lung injury (ALI) is an inflammatory disorder characterized by hypoxemia and diffuse infiltration of neutrophils into the alveolar space. The migration and extravasation of neutrophils is guided through positive guidance cues, such as chemokines. Recent work has identified the neuronal guidance protein netrin-1 to be a negative guidance cue for leukocyte migration and to hold antiinflammatory potential. OBJECTIVES To test the role of pulmonary netrin-1 during ALI. METHODS Pulmonary netrin-1 expression was evaluated during acute inflammation in vitro and in vivo; the netrin-1 promoter was studied using pGL4 luciferase reporter. ALI was induced through LPS inhalation and mechanical ventilation in wild-type, Ntn1(+/-), and A2BAR(-/-) animals. Exogenous netrin-1 was used to evaluate its impact on pulmonary inflammation. MEASUREMENTS AND MAIN RESULTS Wild-type animals demonstrated repression of pulmonary netrin-1 after LPS inhalation. In vitro studies confirmed the repression of netrin-1. Studies in the putative netrin-1 promoter identified a nuclear factor-kappaB-dependent mechanism to be involved in this repression. Ntn1(+/-) animals demonstrated increased inflammatory changes after LPS inhalation compared with Ntn1(+/+) animals. Reconstitution with netrin-1 dampened the infiltration of neutrophils and cytokine production in the alveolar space. This effect was dependent on the adenosine 2b receptor. The importance of netrin-1 for the control of pulmonary inflammation could be corroborated in a model of ventilator-induced lung injury. CONCLUSIONS Pulmonary netrin-1 levels are repressed during ALI. This results in pronounced pulmonary damage, an increased infiltration of neutrophils, and increased pulmonary inflammation. Exogenous netrin-1 significantly dampens the extent of ALI through the adenosine 2B receptor.

The Influence of Epiaortic Ultrasonography on Intraoperative Surgical Management in 6051 Cardiac Surgical Patients

BACKGROUND Intraoperative echocardiography has become a mainstay monitor of cardiac function and a popular diagnostic tool in patients undergoing cardiac procedures. Previous reports suggest that epiaortic ultrasonography (EU) is superior to transesophageal echocardiography and manual palpation in identifying ascending aortic atheroma. Its impact on surgical decision making has not been thoroughly investigated, however. METHODS We retrospectively analyzed the medical records of 6051 consecutive patients who underwent EU of their ascending aorta during cardiac operations between 1996 and 2006 to determine a potential impact on intraoperative surgical decision making. Aortic atheroma was graded according to standard classification. Neurologic complications were evaluated according to the Society of Thoracic Surgeon definition for stroke and transient ischemic attack (TIA). RESULTS The overall impact of EU on surgical decision making was 4.1% and included a change in the technique for inducing cardiac arrest in 1.8%, aortic atherectomy or replacement surgery in 0.8%, requirement for off-pump coronary artery bypass grafting (CABG) in 0.6%, avoidance of aortic cross-clamping and use of ventricular fibrillatory arrest in 0.5%, change in arterial cannulation site in 0.2%, or avoidance of aortic cannulation in 0.2%. The greatest affect of EU was observed in patients undergoing combined CABG with aortic/mitral valve procedures (6.7%). The smallest impact was seen in patients undergoing mitral valve operations (1.4%). Aortic atheroma was more frequent on the anterior aspect of the aorta (n = 171) in patients with a change in surgical plan than on the posterior aspect (n = 78). The overall stroke rate was lower in patients with intraoperative EU compared with all patients undergoing surgical procedures. CONCLUSIONS Epiaortic ultrasonography is a useful technique to detect ascending aortic atheroma, has a significant impact on surgical decision making in more than 4% of cardiac surgical patients, and might result in improved perioperative neurologic outcome.

PMNs facilitate translocation of platelets across human and mouse epithelium and together alter fluid homeostasis via epithelial cell–expressed ecto-NTPDases

Mucosal diseases are often characterized by an inflammatory infiltrate that includes polymorphonuclear leukocytes (PMNs), monocytes, lymphocytes, and platelets. A number of studies have suggested that the interaction of platelets with leukocytes has an essential proinflammatory role. Here, we examined whether platelets migrate across mucosal epithelium, as PMNs are known to do, and whether platelets influence epithelial cell function. Initial studies revealed that human platelets did not efficiently transmigrate across human epithelial cell monolayers. However, in the presence of human PMNs, platelet movement across the epithelium was proportional to the extent of PMN transmigration, and strategies that blocked PMN transmigration diminished platelet movement. Furthermore, platelet-PMN comigration was observed in intestinal tissue derived from human patients with inflammatory bowel disease (IBD). The translocated platelets were found to release large quantities of ATP, which was metabolized to adenosine via a 2-step enzymatic reaction mediated by ecto-nucleotidases, including CD73 and ecto-nucleoside triphosphate diphosphohydrolases (ecto-NTPDases), expressed on the apical membrane of the intestinal epithelial cells. In vitro studies and a mouse model of intestinal inflammation were employed to define a mechanism involving adenosine-mediated induction of electrogenic chloride secretion, with concomitant water movement into the intestinal lumen. These studies demonstrate that ecto-NTPDases are expressed on the apical membrane of epithelial cells and are involved in what we believe to be a previously unappreciated function for platelets in the inflamed intestine, which might promote bacterial clearance under inflammatory conditions.

Vagus nerve controls resolution and pro-resolving mediators of inflammation

Axonal guidance molecule netrin-1 promotes resolution of inflammation, with netrin-1 and resolvin D1 mutually inducing each other’s expression.