Targeting Gαi2 in neutrophils protects from myocardial ischemia reperfusion injury.

Neutrophils are not only involved in immune defense against infection but also contribute to the exacerbation of tissue damage after ischemia and reperfusion. We have previously shown that genetic ablation of regulatory Gαi proteins in mice has both protective and deleterious effects on myocardial ischemia reperfusion injury (mIRI), depending on which isoform is deleted. To deepen and analyze these findings in more detail the contribution of Gαi2 proteins in resident cardiac vs circulating blood cells for mIRI was first studied in bone marrow chimeras. In fact, the absence of Gαi2 in all blood cells reduced the extent of mIRI (22,9% infarct size of area at risk (AAR) Gnai2-/- → wt vs 44.0% wt → wt; p < 0.001) whereas the absence of Gαi2 in non-hematopoietic cells increased the infarct damage (66.5% wt → Gnai2-/- vs 44.0% wt → wt; p < 0.001). Previously we have reported the impact of platelet Gαi2 for mIRI. Here, we show that infarct size was substantially reduced when Gαi2 signaling was either genetically ablated in neutrophils/macrophages using LysM-driven Cre recombinase (AAR: 17.9% Gnai2fl/fl LysM-Cre+/tg vs 42.0% Gnai2fl/fl; p < 0.01) or selectively blocked with specific antibodies directed against Gαi2 (AAR: 19.0% (anti-Gαi2) vs 49.0% (IgG); p < 0.001). In addition, the number of platelet-neutrophil complexes (PNCs) in the infarcted area were reduced in both, genetically modified (PNCs: 18 (Gnai2fl/fl; LysM-Cre+/tg) vs 31 (Gnai2fl/fl); p < 0.001) and in anti-Gαi2 antibody-treated (PNCs: 9 (anti-Gαi2) vs 33 (IgG); p < 0.001) mice. Of note, significant infarct-limiting effects were achieved with a single anti-Gαi2 antibody challenge immediately prior to vessel reperfusion without affecting bleeding time, heart rate or cellular distribution of neutrophils. Finally, anti-Gαi2 antibody treatment also inhibited transendothelial migration of human neutrophils (25,885 (IgG) vs 13,225 (anti-Gαi2) neutrophils; p < 0.001), collectively suggesting that a therapeutic concept of functional Gαi2 inhibition during thrombolysis and reperfusion in patients with myocardial infarction should be further considered.

Early Biologic Initiation After Chronic Pouch Inflammation Diagnosis Does Not Impact Clinical Outcomes.

Approximately 20% of patients with ulcerative colitis (UC) who undergo total proctocolectomy (TPC) with ileal pouch-anal anastomosis (IPAA) develop chronic pouch inflammation (CPI).1 Given the involvement of the small bowel in CPI, there is no consensus on whether it should be managed more like UC or Crohn's disease (CD). Despite limited evidence, biologics are often used for CPI with clinical response rates of 20%-60% with adalimumab and infliximab, 50%-80% with ustekinumab, and 30%-70% with vedolizumab.2,3 Earlier biologic therapy has been associated with greater rates of response and favorable long-term outcomes in patients with CD, however not UC.4-7 The impact of earlier initiation of biologic therapy on CPI clinical outcomes has not been elucidated. The aim of this study was to assess whether timing of biologic initiation relative to CPI diagnosis impacts clinical and endoscopic remission.

Platelets and the Cybernetic Regulation of Ischemic Inflammatory Responses through PNC Formation Regulated by Extracellular Nucleotide Metabolism and Signaling.

Ischemic events are associated with severe inflammation and are here referred to as ischemic inflammatory response (IIR). Recent studies identified the formation of platelet-neutrophil complexes (PNC) as key players in IIR. We investigated the role of extracellular platelet nucleotide signaling in the context of IIR and defined a cybernetic circle, including description of feedback loops. Cybernetic circles seek to integrate different levels of information to understand how biological systems function. Our study specifies the components of the cybernetic system of platelets in IIR and describes the theoretical progression of IIR passing the cybernetic cycle with positive and negative feedback loops based on nucleotide-dependent signaling and functional regulation. The cybernetic components and feedback loops were explored by cytometry, immunohistological staining, functional blocking antibodies, and ADP/ATP measurements. Using several ex vivo and in vivo approaches we confirmed cybernetic parameters, such as controller, sensor, and effector (VASP phosphorylation, P2Y12, ADORAs and GPIIb/IIIa activity), as well as set points (ADP, adenosine) and interfering control and disturbance variables (ischemia). We demonstrate the impact of the regulated platelet-neutrophil complex (PNC) formation in blood and the resulting damage to the affected inflamed tissue. Taken together, extracellular nucleotide signaling, PNC formation, and tissue damage in IIR can be integrated in a controlled cybernetic circle of platelet function, as introduced through this study.

Early Pouchitis Is Associated With Crohn's Disease-like Pouch Inflammation in Patients With Ulcerative Colitis.

BACKGROUND: Despite the initial diagnosis of ulcerative colitis (UC), approximately 10% to 20% of patients develop Crohn's disease-like pouch inflammation (CDLPI) after restorative proctocolectomy (RPC) with ileal pouch anal anastomosis (IPAA). The aim of this study was to evaluate whether early pouchitis, defined as pouchitis within the first year after IPAA, is a predictor of CDLPI. METHODS: This was a retrospective cohort analysis of patients with UC or IBD unclassified (IBDU) who underwent RPC with IPAA at Mount Sinai Hospital between January 2008 and December 2017. The primary outcome was development of CDLPI. Predictors of CDLPI were analyzed via univariable and multivariable Cox regression models. RESULTS: The analytic cohort comprised 412 patients who underwent at least 1 pouchoscopy procedure between 2009 and 2018. Crohn's disease-like pouch inflammation developed in 57 (13.8%) patients a median interval of 2.1 (interquartile range, 1.1-4.3) years after surgery. On univariable analysis, older age at colectomy (hazard ratio [HR], 0.97; 95% CI, 0.95-0.99) was associated with a reduced risk of CDLPI; although early pouchitis (HR, 2.43; 95% CI, 1.32-4.45) and a greater number of pouchitis episodes (HR, 1.38; 95% CI, 1.17-1.63) were associated with an increased risk. On multivariable analysis, early pouchitis (HR, 2.35; 95% CI, 1.27-4.34) was significantly associated with CDLPI. Time to CDLPI was significantly less in patients who developed early pouchitis compared with those who did not (P = .003). CONCLUSION: Early pouchitis is significantly associated with subsequent CDLPI development and may be the first indication of enhanced mucosal immune activation in the pouch.

In a retrospective cohort analysis of 412 patients with ulcerative colitis who underwent restorative proctocolectomy with ileal pouch anal anastomosis, early pouchitis that occurred within the first year after surgery was significantly associated with subsequent Crohn's disease­like pouch inflammation.

Inhibition of CXCR4 and CXCR7 Is Protective in Acute Peritoneal Inflammation.

Our previous studies revealed a pivotal role of the chemokine stromal cell-derived factor (SDF)-1 and its receptors CXCR4 and CXCR7 on migratory behavior of polymorphonuclear granulocytes (PMNs) in pulmonary inflammation. Thereby, the SDF-1-CXCR4/CXCR7-axis was linked with adenosine signaling. However, the role of the SDF-1 receptors CXCR4 and CXCR7 in acute inflammatory peritonitis and peritonitis-related sepsis still remained unknown. The presented study provides new insight on the mechanism of a selective inhibition of CXCR4 (AMD3100) and CXCR7 (CCX771) in two models of peritonitis and peritonitis-related sepsis by injection of zymosan and fecal solution. We observed an increased expression of SDF-1, CXCR4, and CXCR7 in peritoneal tissue and various organs during acute inflammatory peritonitis. Selective inhibition of CXCR4 and CXCR7 reduced PMN accumulation in the peritoneal fluid and infiltration of neutrophils in lung and liver tissue in both models. Both inhibitors had no anti-inflammatory effects in A2B knockout animals (A2B-/-). AMD3100 and CCX771 treatment reduced capillary leakage and increased formation of tight junctions as a marker for microvascular permeability in wild type animals. In contrast, both inhibitors failed to improve capillary leakage in A2B-/- animals, highlighting the impact of the A2B-receptor in SDF-1 mediated signaling. After inflammation, the CXCR4 and CXCR7 antagonist induced an enhanced expression of the protective A2B adenosine receptor and an increased activation of cAMP (cyclic adenosine mono phosphate) response element-binding protein (CREB), as downstream signaling pathway of A2B. The CXCR4- and CXCR7-inhibitor reduced the release of cytokines in wild type animals via decreased intracellular phosphorylation of ERK and NFκB p65. In vitro, CXCR4 and CXCR7 antagonism diminished the chemokine release of human cells and increased cellular integrity by enhancing the expression of tight junctions. These protective effects were linked with functional A2B-receptor signaling, confirming our in vivo data. In conclusion, our study revealed new protective aspects of the pharmacological modulation of the SDF-1-CXCR4/CXCR7-axis during acute peritoneal inflammation in terms of the two hallmarks PMN migration and barrier integrity. Both anti-inflammatory effects were linked with functional adenosine A2B-receptor signaling.

Targeting CD39 Toward Activated Platelets Reduces Systemic Inflammation and Improves Survival in Sepsis: A Preclinical Pilot Study.

OBJECTIVES: Sepsis is associated with a systemic inflammatory reaction, which can result in a life-endangering organ dysfunction. Pro-inflammatory responses during sepsis are characterized by increased activation of leukocytes and platelets, formation of platelet-neutrophil aggregates, and cytokine production. Sequestration of platelet-neutrophil aggregates in the microvasculature contributes to tissue damage during sepsis. At present no effective therapeutic strategy to ameliorate these events is available. In this preclinical pilot study, a novel anti-inflammatory approach was evaluated, which targets nucleoside triphosphate hydrolase activity toward activated platelets via a recombinant fusion protein combining a single-chain antibody against activated glycoprotein IIb/IIIa and the extracellular domain of CD39 (targ-CD39). DESIGN: Experimental animal study and cell culture study. SETTING: University-based experimental laboratory. SUBJECTS: Human dermal microvascular endothelial cells 1, human platelets and neutrophils, and C57BL/6NCrl mice. INTERVENTIONS: Platelet-leukocyte-endothelium interactions were evaluated under inflammatory conditions in vitro and in a murine lipopolysaccharide-induced sepsis model in vivo. The outcome of polymicrobial sepsis was evaluated in a murine cecal ligation and puncture model. To evaluate the anti-inflammatory potential of activated platelet targeted nucleoside triphosphate hydrolase activity, we employed a potato apyrase in vitro and in vivo, as well as targ-CD39 and as a control, nontarg-CD39 in vivo. MEASUREMENTS AND MAIN RESULTS: Under conditions of sepsis, agents with nucleoside triphosphate hydrolase activity decreased platelet-leukocyte-endothelium interaction, transcription of pro-inflammatory cytokines, microvascular platelet-neutrophil aggregate sequestration, activation marker expression on platelets and neutrophils contained in these aggregates, leukocyte extravasation, and organ damage. Targ-CD39 had the strongest effect on these variables and retained hemostasis in contrast to nontarg-CD39 and potato apyrase. Most importantly, targ-CD39 improved survival in the cecal ligation and puncture model to a stronger extent then nontarg-CD39 and potato apyrase. CONCLUSIONS: Targeting nucleoside triphosphate hydrolase activity (CD39) toward activated platelets is a promising new treatment concept to decrease systemic inflammation and mortality of sepsis. This innovative therapeutic approach warrants further development toward clinical application.

Adenosine Receptor Adora2b Plays a Mechanistic Role in the Protective Effect of the Volatile Anesthetic Sevoflurane during Liver Ischemia/Reperfusion.

BACKGROUND: Liver ischemia/reperfusion (IR) injury is characterized by hepatic tissue damage and an inflammatory response. This is accompanied by the formation and vascular sequestration of platelet-neutrophil conjugates (PNCs). Signaling through Adora2b adenosine receptors can provide liver protection. Volatile anesthetics may interact with adenosine receptors. This study investigates potential antiinflammatory effects of the volatile anesthetic sevoflurane during liver IR. METHODS: Experiments were performed ex vivo with human blood and in a liver IR model with wild-type, Adora2a, and Adora2b mice. The effect of sevoflurane on platelet activation, PNC formation and sequestration, cytokine release, and liver damage (alanine aminotransferase release) was analyzed using flow cytometry, luminometry, and immunofluorescence. Adenosine receptor expression in liver tissue was analyzed using immunohistochemistry and real-time polymerase chain reaction. RESULTS: Ex vivo experiments indicate that sevoflurane inhibits platelet and leukocyte activation (n = 5). During liver IR, sevoflurane (2 Vol%) decreased PNC formation 2.4-fold in wild-type (P < 0.05) but not in Adora2b mice (n ≥ 5). Sevoflurane reduced PNC sequestration 1.9-fold (P < 0.05) and alanine aminotransferase release 3.5-fold (P < 0.05) in wild-type but not in Adora2b mice (n = 5). In Adora2a mice, sevoflurane also inhibited PNC formation and cytokine release. Sevoflurane diminished cytokine release (n ≥ 3) and increased Adora2b transcription and expression in liver tissue of wild-types (n = 4). CONCLUSIONS: Our experiments highlight antiinflammatory and tissue-protective properties of sevoflurane during liver IR and reveal a mechanistic role of Adora2b in sevoflurane-associated effects. The targeted use of sevoflurane not only as an anesthetic but also to prevent IR damage is a promising approach in the treatment of critically ill patients.

Inhibition of Plexin C1 Protects Against Hepatic Ischemia-Reperfusion Injury.

OBJECTIVES: Hepatic ischemia-reperfusion injury is a disease pattern that is associated with an acute inflammatory reaction. It is well known that neutrophils play an essential role in the early phase of hepatic ischemia-reperfusion injury and determine the extent of tissue damage. Hepatic ischemia-reperfusion injury can result in organ failure, which is linked to high mortality. Recent data indicate that the neuronal guidance receptor Plexin C1 is involved in the control of the acute inflammatory response and, as such, modulates the transmigration of neutrophils. Hence, we investigated the functional role of Plexin C1 in a mouse model of early hepatic ischemia-reperfusion injury. DESIGN: Animal study. SETTING: University experimental laboratory. SUBJECTS: Wild-type, PLXNC1 and chimeric mice. INTERVENTIONS: Hepatic ischemia-reperfusion injury or sham operation. MEASUREMENTS AND MAIN RESULTS: We found that the functional inhibition of Plexin C1 in wild-type mice treated with an anti-Plexin C1 antibody and a Semaphorin 7A peptide reduced hepatic ischemia-reperfusion injury, as measured by the levels of lactate dehydrogenase, aspartate, and alanine aminotransferase. This reduction in ischemia-reperfusion injury was accompanied by reduced numbers of neutrophils in ischemic hepatic tissue and reduced serum levels of inflammatory cytokines. Experiments using Plexin C1 receptor-deficient (PLXNC1) mice also demonstrated decreased hepatic ischemia-reperfusion injury. Studies of chimeric mice revealed that the hematopoietic Plexin C1 knockout is crucial for reducing the extent of hepatic ischemia-reperfusion injury. CONCLUSIONS: These results describe a role for Plexin C1 during ischemia-reperfusion injury, highlight the role of hematopoietic Plexin C1 in the development of hepatic ischemia-reperfusion injury, and suggest that Plexin C1 is a potential drug target.

Semaphorin 7A Aggravates Pulmonary Inflammation during Lung Injury.

The extent of pulmonary inflammation during lung injury ultimately determines patient outcome. Pulmonary inflammation is initiated by the migration of neutrophils into the alveolar space. Recent work has demonstrated that the guidance protein semaphorin 7A (SEMA7A) influences the migration of neutrophils into hypoxic tissue sites, yet, its role during lung injury is not well understood. Here, we report that the expression of SEMA7A is induced in vitro through pro-inflammatory cytokines. SEMA7A itself induces the production of pro-inflammatory cytokines in endothelial and epithelial cells, enhancing pulmonary inflammation. The induction of SEMA7A facilitates the transendothelial migration of neutrophils. In vivo, animals with deletion of SEMA7A expression showed reduced signs of pulmonary inflammatory changes following lipopolysaccharide challenge. We define here the role of SEMA7A in the development of lung injury and identify a potential pathway to interfere with these detrimental changes. Future anti-inflammatory strategies for the treatment of lung injury might be based on this finding.

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.

The neuroimmune guidance cue netrin-1 controls resolution programs and promotes liver regeneration.

UNLABELLED: Hepatic ischemia/reperfusion (I/R) is a major adverse reaction to liver transplantation, hemorrhagic shock, or resection. Recently, the anti-inflammatory properties of the axonal guidance cue netrin-1 were reported. Here, we demonstrate that netrin-1 also impacts the resolution of inflammation and promotes hepatic repair and regeneration during liver I/R injury. In initial studies, we investigated the induction of netrin-1 and its receptors in murine liver tissues after I/R injury. Hepatic I/R injury was performed in mice with a partial genetic netrin-1 deficiency (Ntn1(+/-) ) or wild-type C57BL/6 treated with exogenous netrin-1 to examine the endogenous and therapeutically administered impact of netrin-1. These investigations were corroborated by studies determining the characteristics of intravascular leukocyte flow, clearance of apoptotic neutrophils (polymorphonuclear cells [PMNs]), production of specialized proresolving lipid mediators (SPMs), generation of specific growth factors contributing to the resolution of inflammation, and liver repair. Hepatic I/R was associated with a significant reduction of netrin-1 transcript and protein in murine liver tissue. Subsequent studies in netrin-1-deficient mice revealed lower efficacies in reducing PMN infiltration, proinflammatory cytokine levels, and hepatic-specific injury enzymes. Conversely, mice treated with exogenous netrin-1 exhibited increased liver protection and repair, reducing neutrophil influx into the injury site, decreasing proinflammatory mediators, increasing efferocytosis of apoptotic PMNs, and stimulating local endogenous biosynthesis of SPMs and the generation of specific growth factors. Finally, genetic studies implicated the A2B adenosine receptor in netrin-1-mediated protection during hepatic I/R injury. CONCLUSION: The present study indicates a previously unrecognized role for netrin-1 in liver protection and its contribution to tissue homeostasis and regeneration.

A phase i study of DMS612, a novel bifunctional alkylating agent.

PURPOSE: DMS612 is a dimethane sulfonate analog with bifunctional alkylating activity and preferential cytotoxicity to human renal cell carcinoma (RCC) in the NCI-60 cell panel. This first-in-human phase I study aimed to determine dose-limiting toxicity (DLT), maximum tolerated dose (MTD), pharmacokinetics (PK), and pharmacodynamics (PD) of DMS612 administered by 10-minute intravenous infusion on days 1, 8, and 15 of an every-28-day schedule. EXPERIMENTAL DESIGN: Patients with advanced solid malignancies were eligible. Enrollment followed a 3+3 design. PKs of DMS612 and metabolites were assessed by mass spectroscopy and PD by γ-H2AX immunofluorescence. RESULTS: A total of 31 patients, including those with colorectal (11), RCC (4), cervical (2), and urothelial (1) cancers, were enrolled. Six dose levels were studied, from 1.5 mg/m(2) to 12 mg/m(2). DLTs of grade 4 neutropenia and prolonged grade 3 thrombocytopenia were observed at 12 mg/m(2). The MTD was determined to be 9 mg/m(2) with a single DLT of grade 4 thrombocytopenia in 1 of 12 patients. Two patients had a confirmed partial response at the 9 mg/m(2) dose level, in renal (1) and cervical (1) cancer. DMS612 was rapidly converted into active metabolites. γ-H2AX immunofluorescence revealed dose-dependent DNA damage in both peripheral blood lymphocytes and scalp hairs. CONCLUSIONS: The MTD of DMS12 on days 1, 8, and 15 every 28 days was 9 mg/m(2). DMS612 appears to be an alkylating agent with unique tissue specificities. Dose-dependent PD signals and two partial responses at the MTD support further evaluation of DMS612 in phase II trials.

The plexin C1 receptor promotes acute inflammation.

Acute inflammation is the pathophysiological basis of important clinical conditions associated with organ failure. The initial inflammatory response is controlled by the chemokine system, yet recent data have indicated that the neuronal guidance cues are significantly involved in the orchestration of this process. Previous work has shown the proinflammatory capacity of the guidance cue semaphorin (Sema) 7a, but the role of one of its target receptors, the plexin C1 (PLXNC1) receptor is to date unknown. We report here that PLXNC1 is expressed outside the nervous system and induced during acute inflammation. PLXNC1(-/-) mice with C57BL/6 background demonstrated decreased inflammatory responses during zymosan A (ZyA)-induced peritonitis. Subsequent in vivo studies revealed altered rolling, adhesion, and transmigration properties of PLXNC1(-/-) leukocytes. Blockade of PLXNC1 was associated with attenuated chemotactic transendothelial migration properties in vitro. Studies in chimeric mice revealed that hematopoietic PLXNC1(-/-) animals demonstrated an attenuated inflammatory response. To probe the therapeutic potential of PLXNC1 we treated C57BL/6 WT mice with an anti-PLXNC1 antibody and a PLXNC1 binding peptide. Both of these interventions significantly dampened ZyA-induced peritonitis. These results implicate an important role of PLXNC1 during an acute inflammatory response and indicate PLXNC1 as a potential target for the control of conditions associated with acute inflammation.

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.

Evaluating the role of Netrin-1 during the early phase of peripheral nerve regeneration using the mouse median nerve model.

BACKGROUND: Less is known about the role of Netrin-1 in the peripheral nervous system. In this study, we evaluated the role of Netrin-1 using the mouse median nerve model for assessment of peripheral nerve regeneration. METHODS: Using real-time PCR and western blot analysis, we examined expression changes of netrin-1 mRNA and Netrin-1 protein after transection and repair of the mouse median nerve in Wild-type animals. We further evaluated histomorphometrical changes as well as the functional recovery of the grasping force after median nerve transection and repair in WT mice and Netrin-1(+/-) heterozygous mice. RESULTS: RT-PCR revealed a 1, 9 fold increase of Netrin-1 mRNA two weeks after nerve transection and repair in the nerve segment distal to the injury site. In Western blot analysis, we could show a high increase of Netrin-1 in the nerve segment distal to the injury site at day 14. Histomorphometrical analysis showed significantly higher cross sectional area and a lower fibre density in heterozygous Netrin-1(+/-) mice. Using the functional grasping test, we could show that peripheral nerve regeneration is significantly diminished in heterozygous Netrin-1(+/-) mice. CONCLUSIONS: Employing the mouse median nerve model in transgenic animals, we demonstrate that Netrin-1 plays an important role during peripheral nerve regeneration.

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.

Bone marrow-derived human mesenchymal stem cells express cardiomyogenic proteins but do not exhibit functional cardiomyogenic differentiation potential.

Despite their paracrine activites, cardiomyogenic differentiation of bone marrow (BM)-derived mesenchymal stem cells (MSCs) is thought to contribute to cardiac regeneration. To systematically evaluate the role of differentiation in MSC-mediated cardiac regeneration, the cardiomyogenic differentiation potential of human MSCs (hMSCs) and murine MSCs (mMSCs) was investigated in vitro and in vivo by inducing cardiomyogenic and noncardiomyogenic differentiation. Untreated hMSCs showed upregulation of cardiac tropopin I, cardiac actin, and myosin light chain mRNA and protein, and treatment of hMSCs with various cardiomyogenic differentiation media led to an enhanced expression of cardiomyogenic genes and proteins; however, no functional cardiomyogenic differentiation of hMSCs was observed. Moreover, co-culturing of hMSCs with cardiomyocytes derived from murine pluripotent cells (mcP19) or with murine fetal cardiomyocytes (mfCMCs) did not result in functional cardiomyogenic differentiation of hMSCs. Despite direct contact to beating mfCMCs, hMSCs could be effectively differentiated into cells of only the adipogenic and osteogenic lineage. After intramyocardial transplantation into a mouse model of myocardial infarction, Sca-1(+) mMSCs migrated to the infarcted area and survived at least 14 days but showed inconsistent evidence of functional cardiomyogenic differentiation. Neither in vitro treatment nor intramyocardial transplantation of MSCs reliably generated MSC-derived cardiomyocytes, indicating that functional cardiomyogenic differentiation of BM-derived MSCs is a rare event and, therefore, may not be the main contributor to cardiac regeneration.

New insights into the molecular pathology of radiation-induced pneumopathy.

BACKGROUND AND PURPOSE: Pneumonitis and fibrosis constitute dose-limiting side effects of thorax or total body irradiation. An improved understanding of the underlying mechanisms is a prerequisite for the development of effective radioprotective strategies. Here we characterized the behavior of resident and immune cells in a murine model of radiation-induced pneumopathy. MATERIALS AND METHODS: Wild type (WT) or RAG-2 deficient C57BL/6 mice received 15 Gray of (hemi)-thorax irradiation in a single dose. Bronchoalveolar lavage fluid (BALF) and lung tissue were collected at defined time points post-irradiation for the determination of apoptosis, microvascular injury, and histological and immunohistochemical analyses. RESULTS: Higher albumin levels and increased apoptosis were detected in the BALF 21 days after irradiation, indicative for delayed damage to resident cells. Irradiation also induced time-dependent changes in the BALF cytokine profile, the recruitment of activated T-cells into the lung and the formation of lipid-loaded resident cells. Lung fibrosis occurred earlier in RAG-2(-/-) mice, which lack mature T and B cells, compared to WT mice. CONCLUSIONS: Thorax irradiation triggers a delayed disturbance of tissue integrity and lipid metabolism in the lung. Activated T-lymphocytes infiltrating the lung tissue upon thorax irradiation participate in the protection of the lung from radiation-induced fibrosis.

Phosphorylation of vasodilator-stimulated phosphoprotein prevents platelet-neutrophil complex formation and dampens myocardial ischemia-reperfusion injury.

BACKGROUND: Recent work has suggested that the formation of platelet-neutrophil complexes (PNCs) aggravates the severity of inflammatory tissue injury. Given the importance of vasodilator-stimulated phosphoprotein (VASP) for platelet function, we pursued the role of VASP on the formation of PNCs and its impact on the extent of myocardial ischemia-reperfusion (IR) injury. METHODS AND RESULTS: In initial in vitro studies we found that neutrophils facilitated the movement of platelets across endothelial monolayers. Phosphorylation of VASP reduced the formation of PNCs and transendothelial movement of PNCs. During myocardial IR injury, VASP(-/-) animals demonstrated reduced intravascular formation of PNCs and reduced presence of PNCs within the ischemic myocardial tissue. This was associated with reduced IR injury. Studies using platelet transfer and bone marrow chimeric animals showed that hematopoietic VASP expression was crucial for the intravascular formation of PNCs the presence of PNCs within ischemic myocardial tissue and the extent of myocardial IR injury. Furthermore, phosphorylation of VASP on Ser153 or Ser235 reduced intravascular PNC formation and presence of PNCs within ischemic myocardial tissue. This finding was associated with reduced myocardial IR injury. CONCLUSION: Previously unappreciated, the phosphorylation of VASP performs a key function for the formation of PNCs that is crucially important for the extent of myocardial IR injury.

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 Ser(153) through Prostaglandin E1 or on Ser(235) 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.