Julio C. Morote-Garcia

Crucial Role for Ecto-5′-Nucleotidase (CD73) in Vascular Leakage during Hypoxia

Extracellular adenosine has been widely implicated in adaptive responses to hypoxia. The generation of extracellular adenosine involves phosphohydrolysis of adenine nucleotide intermediates, and is regulated by the terminal enzymatic step catalyzed by ecto-5′-nucleotidase (CD73). Guided by previous work indicating that hypoxia-induced vascular leakage is, at least in part, controlled by adenosine, we generated mice with a targeted disruption of the third coding exon of Cd73 to test the hypothesis that CD73-generated extracellular adenosine functions in an innate protective pathway for hypoxia-induced vascular leakage. Cd73 −/− mice bred and gained weight normally, and appeared to have an intact immune system. However, vascular leakage was significantly increased in multiple organs, and after subjection to normobaric hypoxia (8% O2), Cd73 −/− mice manifested fulminant vascular leakage, particularly prevalent in the lung. Histological examination of lungs from hypoxic Cd73 −/− mice revealed perivascular interstitial edema associated with inflammatory infiltrates surrounding larger pulmonary vessels. Vascular leakage secondary to hypoxia was reversed in part by adenosine receptor agonists or reconstitution with soluble 5′-nucleotidase. Together, our studies identify CD73 as a critical mediator of vascular leakage in vivo.

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.

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.

Physiological Roles of Vascular Nucleoside Transporters

Nucleoside transporters (NTs) comprise 2 widely expressed families, the equilibrative nucleoside transporters (diffusion-limited channels) and concentrative nucleoside transporters (sodium-dependent transporters). Because of their anatomic position at the blood-tissue interface, vascular NTs are in an ideal position to influence vascular nucleoside levels, particularly adenosine, which among others plays an important role in tissue protection during acute injury. For example, endothelial NTs contribute to preserving the vascular integrity during conditions of limited oxygen availability (hypoxia). Indeed, hypoxia-inducible factor-1-dependent repression of NTs results in enhanced extracellular adenosine signaling and thus attenuates hypoxia-associated increases in vascular leakage. In addition, vascular NTs also contribute to cardiac ischemic preconditioning, coronary vasodilation, and inhibition of platelet aggregation. Moreover, vascular nucleoside uptake via NTs is important for nucleoside recovery, particularly in cells lacking de novo nucleotide synthesis pathways (erythrocytes, leukocytes). Taken together, vascular NTs are critical in modulating adenosine-mediated responses during conditions such as inflammation or hypoxia.

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.

Endothelial Semaphorin 7A promotes neutrophil migration during hypoxia

Recent studies identified basic biological principles that are shared by the immune and the nervous system. One of these analogies applies to the orchestration of cellular migration where guidance proteins that serve as a stop signal for axonal migration can also serve as a stop signal for the migration of immune-competent cells. The control of leukocyte migration is of key interest during conditions associated with inflammatory tissue changes such as tissue hypoxia or hypoxic inflammation. Semaphorins are members of these axon guidance molecules. Previously unknown, we report here the expression and induction of semaphorin 7A (SEMA7A) on endothelium through hypoxia-inducible factor 1α during hypoxia. This induction of SEMA7A translates into increased transmigration of polymorphonuclear neutrophil granulocytes across endothelial cells. Extension of these findings demonstrated an attenuated extravasation of polymorphonuclear neutrophil granulocytes in Sema7a-deficient mice from the vasculature during hypoxia. Studies using chimeric animals identified the expression of Sema7A on nonhematopoietic tissue to be the underlying cause of the observed results. Taken together, our findings demonstrate that neuronal guidance proteins do not only serve as a stop signal for leukocyte migration but also can propagate the extravasation of leukocytes from the vascular space. Future anti-inflammatory strategies might be based on this finding.

Inflammation-associated repression of vasodilator-stimulated phosphoprotein (VASP) reduces alveolar-capillary barrier function during acute lung injury

Acute lung injury (ALI) is an inflammatory disorder associated with reduced alveolar‐capillary barrier function, increased pulmonary vascular permeability, and infiltration of leukocytes into the alveolar space. Pulmonary function might be compromised, its most severe form being the acute respiratory distress syndrome. A protein central to physiological barrier properties is vasodilator‐stimulated phosphoprotein (VASP). Given the fact that VASP expression is reduced during periods of cellular hypoxia, we investigated the role of VASP during ALI. Initial studies revealed reduced VASP expressional levels through cyto‐kines in vitro. Studies in the putative human VASP promoter identified NF‐κB as a key regulator of VASP transcription. This VASP repression results in increased paracellular permeability and migration of neutrophils in vitro. In a model of LPS‐induced ALI, VASP −/− mice demonstrated increased pulmonary damage compared with wild‐type animals. These findings were confirmed in a second model of ventilator‐induced lung injury. Studies employing bone marrow chimeric animals identified tissue‐specific repression of VASP as the underlying cause of decreased barrier properties of the alveolar‐capillary barrier during ALI. Taken together these studies identify tissue‐specific VASP as a central protein in the control of the alveolar‐capillary barrier properties during ALI.—Henes, J., Schmit, M. A., Morote‐Garcia, J. C., Mirakaj, V., Kohler, D., Glover, L., Eldh, T., Walter, U., Karhausen, J., Colgan, S. P., Rosenberger, P. Inflammation‐associated repression of vasodilator‐stimulated phosphoprotein (VASP) reduces alveolar‐capillary barrier function during acute lung injury. FASEB J. 23, 4244‐4255 (2009). www.fasebj.org

Phosphorylation of Vasodilator-Stimulated Phosphoprotein (VASP) Dampens Hepatic Ischemia-Reperfusion Injury

Recent work has demonstrated that the formation of platelet neutrophil complexes (PNCs) affects inflammatory tissue injury. Vasodilator-stimulated phosphoprotein (VASP) is crucially involved into the control of PNC formation and myocardial reperfusion injury. Given the clinical importance of hepatic IR injury we pursued the role of VASP during hepatic ischemia followed by reperfusion. We report here that VASP −/− animals demonstrate reduced hepatic IR injury compared to wildtype (WT) controls. This correlated with serum levels of lactate dehydrogenase (LDH), aspartate (AST) and alanine (ALT) aminotransferase and the presence of PNCs within ischemic hepatic tissue and could be confirmed using repression of VASP through siRNA. In studies employing bone marrow chimeric mice we identified hematopoietic VASP to be of crucial importance for the extent of hepatic injury. Phosphorylation of VASP on Ser153 through Prostaglandin E1 or on Ser235 through atrial natriuretic peptide resulted in a significant reduction of hepatic IR injury. This was associated with a reduced presence of PNCs in ischemic hepatic tissue. Taken together, these studies identified VASP and VASP phosphorylation as crucial target for future hepatoprotective strategies.

Repression of the Equilibrative Nucleoside Transporters Dampens Inflammatory Lung Injury

Acute lung injury (ALI) is a devastating disorder of the lung that is characterized by hypoxemia, overwhelming pulmonary inflammation, and a high mortality in the critically ill. Adenosine has been implicated as an anti-inflammatory signaling molecule, and previous studies showed that extracellular adenosine concentrations are increased in inflamed tissues. Adenosine signaling is terminated by the uptake of adenosine from the extracellular into the intracellular compartment via equilibrative nucleoside transporters (ENTs). However, their role in controlling adenosine signaling during pulmonary inflammation remains unknown. After inflammatory in vitro experiments, we observed a repression of ENT1 and ENT2 that was associated with an attenuation of extracellular adenosine uptake. Experiments using short, interfering RNA silencing confirmed a significant contribution of ENT repression in elevating extracellular adenosine concentrations during inflammation. Furthermore, an examination of the ENT2 promoter implicated NF-κB as a key regulator for the observed ENT repression. Additional in vivo experiments using a murine model of inflammatory lung injury showed that the pharmacological inhibition of ENT1 and ENT2 resulted in improved pulmonary barrier function and reduced signs of acute inflammation of the lung. Whereas experiments on Ent1(-/-) or Ent2(-/-) mice revealed lung protection in LPS-induced lung injury, an examination of bone marrow chimeras for ENTs pointed to the nonhematopoetic expression of ENTs as the underlying cause of dampened pulmonary inflammation during ALI. Taken together, these findings reveal the transcriptional repression of ENTs as an innate protective response during acute pulmonary inflammation. The inhibition of ENTs could be pursued as a therapeutic option to ameliorate inflammatory lung injury.

Inhibition of Adenosine Kinase Attenuates Acute Lung Injury.

Objectives:Extracellular adenosine has tissue-protective potential in several conditions. Adenosine levels are regulated by a close interplay between nucleoside transporters and adenosine kinase. On the basis of the evidence of the role of adenosine kinase in regulating adenosine levels during hypoxia, we evaluated the effect of adenosine kinase on lung injury. Furthermore, we tested the influence of a pharmacologic approach to blocking adenosine kinase on the extent of lung injury. Design:Prospective experimental animal study. Setting:University-based research laboratory. Subjects:In vitro cell lines, wild-type and adenosine kinase+/– mice. Interventions:We tested the expression of adenosine kinase during inflammatory stimulation in vitro and in a model of lipopolysaccharide inhalation in vivo. Studies using the adenosine kinase promoter were performed in vitro. Wild-type and adenosine kinase+/– mice were subjected to lipopolysaccharide inhalation. Pharmacologic inhibition of adenosine kinase was performed in vitro, and its effect on adenosine uptake was evaluated. The pharmacologic inhibition was also performed in vivo, and the effect on lung injury was assessed. Measurements and Main Results:We observed the repression of adenosine kinase by proinflammatory cytokines and found a significant influence of nuclear factor kappa-light-chain-enhancer of activated B-cells on regulation of the adenosine kinase promoter. Mice with endogenous adenosine kinase repression (adenosine kinase+/–) showed reduced infiltration of leukocytes into the alveolar space, decreased total protein and myeloperoxidase levels, and lower cytokine levels in the alveolar lavage fluid. The inhibition of adenosine kinase by 5-iodotubercidin increased the extracellular adenosine levels in vitro, diminished the transmigration of neutrophils, and improved the epithelial barrier function. The inhibition of adenosine kinase in vivo showed protective properties, reducing the extent of pulmonary inflammation during lung injury. Conclusions:Taken together, these data show that adenosine kinase is a valuable target for reducing the inflammatory changes associated with lung injury and should be pursued as a therapeutic option.